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R through activating the inflammasome and inducing pyroptosis
International Immunopharmacology 80 (2020) 106183
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NLRP6 expressed in astrocytes aggravates neurons injury after OGD/R through activating the inflammasome and inducing pyroptosis ⁎
Jinyan Zhanga,1, Ning Jianga,1, Luyu Zhangb, Changchang Mengc, Jing Zhaoc,d, , Jingxian Wua,d,
T ⁎
a
Department of Pathology, Chongqing Medical University, Chongqing, People’s Republic of China Department of Molecular Medicine, Chongqing Medical University, Chongqing, People’s Republic of China c Department of Pathophysiology, Chongqing Medical University, Chongqing, People’s Republic of China d Institute of Neuroscience, Chongqing Medical University, Chongqing, People’s Republic of China b
A R T I C LE I N FO
A B S T R A C T
Keywords: NLRP6 Stroke OGD/R Inflammation Pyroptosis Inflammasomes
NLRP6, the nucleotide oligomerization domain-like receptor family pyrin domain containing 6, has a substantiable effect on inflammation and host defense against microorganisms. In our previous study, NLRP6 promotes inflammation after cerebral I/R injury in a MCAO model. However, the effect of NLRP6 in different nerve cells subjected to OGD/R needs to be further understood. Here, evidence shows that the expression of NLRP6 is increased in different nerve cells subjected to OGD/R, and mainly expressed in astrocytes. NLRP6 may up-regulate inflammation factors (IL-1β, Il-8) via the form of inflammasomes in astrocytes after OGD/R. Then, primary neuron-astrocyte co-culture model under OGD/R in vitro was performed, and we found that NLRP6 decreased the neurons viability and aggravated apoptosis of neurons. Mechanically, NLRP6 could induce pyroptosis to regulate the survival of neurons through activating caspase-1.
1. Introduction Stroke is the second primary incentive of death of human as well as a crucial source of being disable. About 87% stroke patients are ischemic [1]. Recent evidence has indicated that inflammasomes are key intracellular receptors and sensors in innate immune system responsible for the initiation of sterile inflammation in CNS (central nervous system) diseases, for instance, traumatic brain injury [2], ischemic stroke [3], and ICH (intracerebral hemorrhage) [4]. Inflammasomes are crucial elements in stroke pathology [5] and necessary for inflammatory cytokine maturation [6]. NLRs (the nucleotide oligomerization domain-like receptor family) are currently known to assemble inflammasome complexes and to recognize pathogen associated molecular patterns (PAMPs). NLRs can also recruit pro-caspase-1 to form inflammasomes by binding to the adapter protein ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), being critical to caspase-1 initialization and subsequent secretion of IL-18 and IL-1β [7]. The previous study reveals that NLRP3 inflammasome hinderance played a neuroprotective role in cerebral I/R injury [8,9]. Besides, GSK-3β (glycogen synthase kinase 3β) inhibition-induced protection effect in cerebral I/R injury is partially attributed to the inhibition of NLRP3 inflammasomes [10]. In
addition, ever increasing evidence has uncovered that inflammasomemediated pyroptosis is engaged in the pathological progression of I/R injury in cerebral tissues. Caspase-1 is actuated on inflammasomes. Caspase-1 activation promotes pro- IL-1β and pro-IL-18 in activation, which is considered as the rate limiting step in inflammation. Caspase-1 activation also cleaves the gasdermins (GSMDs), which is responsible for the pore formation in cell membrane and subsequent pyroptosis [11,12]. Recently one study showed that blockage of NLRP3-induced pyroptosis contributed to the neuroprotective effects exerted by hispidulin via the AMPK/GSK-3β (AMP-activated protein kinase/glycogen synthase kinase 3β) signaling pathway [13]. From the above, inflammasome-mediated inflammation and pyroptosis play a key role in I/R injury of the brain. NLRP6, a member of the NLRs family, which is similar to other NLRs, could participate in inflammasome formation [14]. Our previous study firstly revealed that the level of NLRP6 was increased in a rat model of MCAO and inhibition of NLRP6 alleviated inflammation [15]. Additionally, it has been demonstrated that pyroptosis of gingival fibroblasts can be triggered by NLRP6 via activating caspase-1 [16]. However, it’s interesting to find that NLRP6 could be independent of the initialization of the inflammasome and have an important effect on protecting mice against colitis by reprogramming the gut microbiota
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Corresponding authors at: Department of Pathology, Chongqing Medical University, Chongqing, People’s Republic of China. E-mail addresses: [email protected] (J. Zhao), [email protected] (J. Wu). 1 These authors equally contributed to the work. https://doi.org/10.1016/j.intimp.2019.106183 Received 2 October 2019; Received in revised form 21 December 2019; Accepted 31 December 2019 1567-5769/ © 2020 Elsevier B.V. All rights reserved.
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and synthesized by GenePharma Corporation (Shanghai, China). In accordance with the NLRP6 gene of rat, the siRNA sequences (sense: 5′-GCAGACGAAUUGCCUACUUTT-3′; anti-sense: 5′-AAGUAGGCAAUU CGUCUGCTT-3′), and the control siRNA (sense: 5′-UUCUCCGAACGU GUCACGUTT-3′; antisense: 5′-ACGUGACACGUUCGGAGAATT-3′) were designed. Lipofectamine 2000 (Invitrogen) was used for siRNA transfection. Western blot was employed to examine the level of NLRP6.
[17]. Another study also showed that NLRP6 promoted the recovery of peripheral nerve injury by inhibiting the inflammatory response independent of inflammasomes [18]. In brief, these studies revealed that NLRP6 was associated with inflammation injury, which may or may not be dependent on inflammasomes. However, the function of NLRP6 in ischemia stroke is largely unknown, and its further mechanism still needs full investigation. Therefore, we hypothesized that NLRP6 may influence inflammation and pyroptosis after OGD/R in neural cells through inflammasome formation, and play an important role in the cerebral I/R injury.
2.5. Lentiviral transfection of NLRP6 overexpression NLRP6 overexpression lentivirus was purchased from GenePharma Corporation (Shanghai, China). No inserts in scramble lentivirus. Astrocytes were transfected with overexpression or scramble lentivirus in the medium to achieve a MOI (multiplicity of infection) of 15. GFP (green fluorescent protein)-positive cells were overexpressed as well as scramble cells. RFP (red fluorescent protein)-positive cells were astrocytes. The fluorescence microscope was used to confirm GFP and RFP cells. Western blot was employed to determine sustained NLRP6 overexpression efficiency.
2. Materials and methods 2.1. Cell culture We isolated astrocytes from Sprague-Dawley (SD) rats (aged one day), and neurons were isolated from SD rat embryos (gestational 17 days). Cell line of microglia (hapi) was donated by Institute of Neuroscience of Chongqing Medical University. Further details will be given afterwards.
2.6. q-PCR arrays 2.2. Co-culture Total RNA was isolated from cells using TRIzol reagent (Invitrogen) following manufacturer manual, and RNA concentration was measured by NanoDrop 8000 (Thermo Fisher Scientific). Reverse transcription was performed using a GoScript reverse transcription system (Promega) according to the manufacturer’s protocol. Then, q-PCR was performed using TaKaRa SYBR Premix Ex Taq II (Tli RNase H Plus) (TaKaRa Biotechnology) with a CFX96 Touch™ Real-Time PCR Detection System. Primer sequences were used for the amplification of NLRP6 (5′-CCGC ATCGTCTACTGTTCATCCTG-3′; 3′-GCACTCTCAAGCCACTCG-TAGC-5′) and GAPDH (5′-GACATGCCGCCTGGAGAAAC −3′; 3′-AGCC CAGGAT GCCCTTTAGT-5′). GAPDH was taken as an endogenous control.
Primary astrocytes were prepared by using of Park and colleagues [19] and plated in 75-cm2 T-flasks. After 2–3 weeks of culture, microglia and oligodendrocytes which growing above the astrocytes layer were detached from the flasks by mild shaking. The remaining adherent astrocytes were detached with trypsin and plated onto 14-mm poly-Llysine-coated glass coverslips in 24-well plates. When astrocytes were adhered on the glass coverslips in 24-well plates and almost reached 70–80% confluence, primary neurons were prepared by using previously published methods [20]. Primary neurons were plated directly on the plastic surface of 24-well plates coated with poly-L-lysine. Neuron purity (> 90%) was assessed using the neuron-specific marker, NeuN. Non-contact neuron-astrocyte co-cultures were established as previously described with modification [21]. The co-culture model is shown in the figure below. In brief, primary neurons were adhered on the plastic surface in 24-well platea, they were incubated with primary astrocytes which adhered on the glass coverslips on paraffin columns in the culture medium containing 2% B27 and 1% Pen/Strep. Here, paraffin columns which were not harmful to co-culture were used to separate neurons and astrocytes.
2.7. Western blot analysis A RIPA (radioimmunoprecipitation assay) lysis buffer (provided by Beyotime) accompanied with protease inhibitor (Roche, Basel, Switzerland) was utilized to separate total protein from the cultured cell. Protein electrophoresis was conducted on an 8–15% SDS/polyacrylamide gel (SDS/PAGE) and protein samples were then transferred to the PVDF membrane, which was then blocked by TBS-Tween buffer (pH 7.5; 5% nonfat milk, 20 mM Tris-HCL, 0.05% Tween-20, and 150 mM NaCl) for 2 h under room temperature. The membrane was subjected to incubation overnight at 4C. Following contents were used during incubation: primary antibodies for NALP6 (Thermo Fisher Scientific, USA); IL-18 (Protein tech, China); IL-1β (Affinity, USA); caspase-1 (Cell Signaling Technology, USA); ASC (Santa Cruz Biotechnology, USA), GSDMD-NT (Abcam, USA) and β-actin (Proteintech, China). TBS-T washing was performed three times with an interval of 10 min for the membrane, which was then incubated with the secondary antibody (anti-rabbit or anti-rat IgG) for 2 h under room temperature, followed by TBS-T washing for three times with an interval of 10 min. Enhanced chemiluminescence (ECL) reagent was added following the manufacturer manual (Bio-Rad). Afterwards, X-ray was used to detect the signals from the ECL.
2.3. OGD/R To stimulate ischemic-like circumstance outside in vitro, cell culture was subjected to OGD (oxygen-glucose deprivation) and subsequently transferred into the original medium with oxygen (95% air, 5% CO2) as well as glucose for 24 h. Anaerobic chamber (Thermo Scientific) with gas mixtures (1% O2, 5% CO2 as well as 94% N2) and deoxygenated glucose-free Hanks' Balanced Salt Solution (provided by Invitrogen) was used for OGD.
2.8. Immunofluorescence staining Astrocytes, microglia and neuronal cells were cultured on coverslips and exposed to OGD for 0, 1, 2, 4, or 6 h, respectively. Then the injured cultured cells were reperfused for twenty-four hours and fixed using paraformaldehyde (4%) in PBS. The coverslip was washed twice with PBS, followed by incubation with a primary antibody at four centigrade degree overnight. Then, the coverslip was incubated one hour with a fluorescein-labelled secondary antibody. DAPI was used to stain cellular
2.4. SiRNA transfection of NLRP6 Small interfering RNA (siRNA) sequences of NLRP6 were designed 2
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nuclei. Cells were imagined with an Olympus confocal fluorescence microscope.
3.2. The effect of NLPR6 intervention on inflammatory factors The results above showed that NLRP6 increased in astrocytes after OGD/R. And NLRP6 was related to inflammation, so this experiment intended to analyze the relationship between NLRP6 and inflammation in astrocytes after OGD/R. We then constructed siRNA of NLRP6 and NLRPR overexpression lentivirus to transfect astrocytes subjected to OGD/R. Western blot and ELISA were applied to measure levels of IL18, IL-1β, and Caspase-1. NLRP6 silence decreased the protein expression of the inflammatory factors IL-18, IL-1β, and Caspase-1, while NLRP6 overexpression induced a significant expression of IL-18, IL-1β, and Caspase-1 (Fig. 2A, B, C, D). Meanwhile, the data of ELISA were consistent with the preceding results (Fig. 2F and G). The results from this part of experiments revealed that NLRP6 may play a pro-inflammatory role in astrocytes after OGD/R.
2.9. Cell viability Cell Counting Kit-8 (CCK-8, Kumamoto, Japan) was implemented for neuronal viability assay following manufacturer manual. In brief, CCK-8 solutions were introduced to each coverslip containing OGD/R cells and the absorbance at 450 nm was examined by a microplate reader. 2.10. ELISA (enzyme-linked immunosorbent assay) Expression levels of IL-1B and IL-18 in the cell supernatants were detected using the relative rat ELISA Kit (Dakewe, China). 2.11. Co-immunoprecipitation
3.3. The effect of NLRP6 in neurons co-culture with astrocytes following OGD/R
Cultured primary astrocytes were harvested and total protein was isolated. Co-immunoprecipitation (Co-IP) was conducted following manufacturer's instruction. Anti-ASC (Santa Cruz Biotechnology, USA), anti-NLRP6 antibody (Bioss, China), and protein A/G agarose (Beyotime, China) were used. After incubation the cells with beads, they underwent centrifugation at 2,500g for 5 min, a total of 5 times. Co-IP was examined by Western blotting.
To determine the role of NLRP6 in primary neurons with primary astrocytes co-cultured in 24-well plates, we used NLRP6 siRNA and NLRP6 overexpressing lentivirus following OGD/R, and then the viability and apoptosis of neurons were detected by CCK8 assay and FCM (flow cytometry). Results showed that NLRP6 knockdown significantly caused a reduced percentage of neuronal apoptosis and promoted neuronal viability, whereas NLRP6 overexpression emerged the opposite effects (Fig. 3A, B, C). These data indicated that NLRP6 may exert a non-protective function in neurons-astrocytes co-culture model after OGD/R injury.
2.12. Flow cytometry After the cells subjected to OGD/R, removing medium, and wash the cells twice with PBS slowly. Removing the PBS, adding 1 mL of 0.125% trypsin in every well and placing the plate to stand at 37 °C incubator for 20 min, the plate with mild shaking every 5 min to accelerate cell shedding. Then, adding 1 mL neuronal culture medium to stop digestion. Pipetting the cells off the plate, and transferring them to centrifuge tube. Pelleting the cells by centrifugation at 1000g for 5 min. Removing the medium and resuspending the cells in PBS. Counting and adjusting the cells to 1 × 106 cells/mL in PBS. Cells are labeled by PI (Propidium Iodide) and FITC-coupled annexin V (annexin V-FITC) double staining for detection of neurons apoptosis. To specifically block caspase-1, AcYVAD-cmk (APExBIO, USA) was used. Caspase-1-FITC (Immunochemistry Technologies, USA) was used to examine the activity of caspase-1. The apoptosis and pyroptosis were identified by flow cytometry (CytoFLEX, USA).
3.4. NLRP6 binds to ASC, then activating caspase-1 to induce the release of inflammatory factors To identify whether NLRP6 activates the inflammasome pathway, co-immunoprecipitation assay was conducted. NLRP6-ASC was more effective in co-precipitating of astrocytes infected via NLRP6 overexpression lentivirus while NLRP6-ASC was less co-precipitated in astrocytes infected by NLRP6 siRNA (Fig. 4A). Lentiviral overexpression of NLRP6 promoted pro-caspase-1 cleavage into activation form in astrocytes, which was inhibited by Ac-YVAD-cmk (caspase-1 inhibitor) (Fig. 4B). The effects of NLRP6 overexpression on the secretion of IL-18 and IL-1β were detected by Western blotting analysis. The analysis exhibit that lentiviral overexpression of NLRP6 elevated the protein expression levels of IL-18 and IL-1β, which was blocked by caspase-1 inhibitor Ac-YVAD-cmk (Fig. 4B). These findings suggested that the increase of IL-18 and IL-1β secretion was attributed to NLRP6-induced caspase-1 activation in astrocytes.
2.13. Statistical analysis Values were described as mean ± SEM, and one-way ANOVA, twoway ANOVA or t test with GraphPad Prism 7.0 software was employed for statistical analysis. P < 0.05 meant significant difference. 3. Results
3.5. NLRP6 mediates pyroptosis by activating Caspase-1 in neurons coculture with astrocytes after OGD/R
3.1. The expression of NLRP6 in neurons, astrocytes and microglia cells To further discuss the relationship between NLRP6 and pyroptosis after OGD/R models, we firstly detected the level of GSDMD-NT with NLRP6 knockdown or overexpression in neurons-astrocytes co-cultured model after OGD 4 h. Then, we identified whether NLRP6 induced pyroptosis through activating Caspase-1. NLRP6 silence decreased the protein levels of the pyroptosis related factors GSDMD-NT, while NLRP6 overexpression induced a significant expression of GSDMD-NT (Fig. 5A, C, D). In addition, Ac-YVAD-CMK (Caspase-1 inhibitor) can rescue NLRP6-mediated pyroptosis in co-cultured cells after OGD/R (OGD/R + LV-NLRP6 group vs OGD/R + LV-NLRP6 + Ac-YVAD-CMK group, Fig. 5B, E, F), indicating that NLRP6 mediated pyroptosis by activating Caspase-1. As illustrated in Fig. 5G and H, the trend of FCM is consistent with the results of Western blot.
To investigate the effect of NLRP6 in OGD/R models, we first used immunofluorescence to evaluate the expression of NLRP6 in primary neurons, astrocytes and microglia cells. As labelled by immunofluorescence staining for NLRP6 and neuronal marker NeuN, astrocytic marker GFAP, and microglial maker Iba-1, and we found that NLRP6 was mainly expressed in astrocytes and reached the peak at 4 h after OGD/R (Fig. 1B), but little expressed in neurons and microglia (Fig. 1A and C). Then, western blot was utilized to investigate the expression of NLRP6 in cultured primary astrocytes after OGD/R and found that NLRP6 protein was markedly increased in the OGD/R, with the peak expression of NLRP6 at 4 h after OGD/R (Fig. 1D and E). The mRNA level of NLRP6 was consistent with results of western blots (Fig. 1F). 3
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Fig. 1. The expression of NLRP6 in neuron, astrocyte and microglia cells. (A) Immunofluorescence staining for NeuN and NLRP6; (B) Immunofluorescence staining for GFAP and NLRP6; (C) Immunofluorescence staining for IBA1 and NLRP6; (D) Western blot of NLRP6 expression in sham, 1, 2, 4, 6 h after OGD in astrocytes; (E) Relative quantification of NLRP6. (F) The mRNA level of NLRP6 in sham, 1, 2, 4, 6 h after OGD in astrocytes. Data are presented as means ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001 versus sham group. NeuN: neuron-specific nuclear protein; GFAP: glial fibrillary acidic protein; Iba-1: ionized calcium binding adapter molecule 1.
4. Discussion
whether it is dependent on the inflammasome-induced by NLRP6. In our previous study, we found that NLRP6 promoted the release of inflammation factors (IL-1β, IL-8) after I/R injury in the brain tissues of MCAO models [15]. Our investigation showed that NLRP6 also upregulated inflammation factors (IL-1β, IL-18) in astrocytes after OGD/R models, which is consistent with our previous findings. In addition, our experimental data demonstrated that NLRP6 interacted with ASC, and inhibiting caspase-1 could decrease the release of inflammatory factors. These results indicated that similar to other NLRs, NLRP6 played a proinflammation effect in OGD/R models in astrocytes dependent on inflammasomes. In recent years, many researches focus on an inflammatory cell death mode-pyroptosis. Pyroptosis is an inflammatory form of programmed cell death, which is characterized by activation of caspase-1 followed by IL-18 and IL-1β, and cellular extravasation [28]. Within the canonical pathway, pyroptosis is characterized by initialization of inflammasomes, and then activated caspases-1 cleaves GSDMD to generate GSDMD-NT, which further induces membrane pore formation and mediates proptosis [29]. Pyroptosis has been identified to be activated both in vivo and in vitro stroke models [30]. More importantly, pyroptotic neuronal and glial cell death trigged by stroke, contributes to neurological deficits, neuroinflammation and the imbalance of homeostasis in the CNS [31]. A recent report shows that pyroptosis of gingival fibroblasts could be attributed to NLRP6 induced activation of caspase-1 [16]. In our preliminary experiment, we found that NLRP6 may influence the survival of neurons, because the increased expression of NLRP6 significantly decreased the neurons vitality and aggravated neurons apoptosis. In the meantime, we identified that NLRP6 could trigger GSDMD-NT in primary neurons-astrocytes co-cultured model after OGD/R. Furthermore, we added Ac-YVAD-CMK, a peptide inhibitor of caspase-1, and it was found that inhibition of caspase-1 could
For the first time, we found that OGD/R elevated the protein expression level of NLRP6 and NLRP6 was largely colocalized with GFAP in the primary cultured astrocytes. We also illustrated that NLRP6 enhanced the release of inflammatory factors via the form of inflammasomes. In addition, intervention of NLRP6 may influence the viability and apoptosis of neurons. More importantly, it is the originality that NLRP6 regulated pyroptosis by activating Caspase-1 in neurons-astrocytes co-cultured model after OGD/R. Previous reports have indicated that NLRP6 is expressed in different tissues, including the kidney, liver, lung, stomach, pancreas, spleen and small and large intestines. Due to the relative high expression of NLRP6 in the intestines, studies of NLRP6 function largely focus on the intestines [22–24]. Recently, increasing evidence suggests that NLRs critically affect ischemic stroke pathogenesis, development and progression [7]. Moreover, blockage of NLRP3 inflammasomes at different cellular levels may provide some therapeutic strategies against neurological deterioration in ischemic stroke [25]. While it remains unclear about the effects of I/R injury in brain tissues on the expression and function of NLRP6 in the CNS. For the first time, our results showed that NLRP6 was principally expressed in the astrocytes and the resulting expression was increased after OGD/R. The pathophysiology of cerebral I/R injury is complex, which has multiple damage and cascade reactions, including inflammation, oxidative stress and cell death [26]. NLRP6, called PYPAF5 initialy, has been implicated to activate caspase1 in the inflammasome signaling, which correspondently activates IL1β and IL-18 [27]. However, emerging studies show that NLRP6 protects against colitis and colitis-related colorectal cancer via inflammasome-independent mechanisms [17]. Thus, it is very interesting to investigate the impact of NLRP6 on the I/R injury in the brain tissues, and 4
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Fig. 2. The effect of NLPR6 intervention on inflammation factors. The cultured primary astrocytes were transfected with NLRP6 interference lentivirus or overexpression lentivirus, following OGD(4 h)/R(24 h). (A) Western blot was used to measure the protein levels of NLRP6, cleaved–caspase-1, IL-1β and IL-18. Relative quantification of NLRP6 (B), cleaved–caspase-1 (C), IL-1β(D) and IL-18 (E). (F, G) ELISA assay was used to measure IL-1β and IL-18 levels in cell supernatants. Data are presented as means ± SEM. ** P < 0.01, versus sham group; # P < 0.05, ## P < 0.01, versus NC group.
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Fig. 3. The effect of NLRP6 in neurons co-cultured with astrocytes following OGD/R. The cultured primary astrocytes were transfected with NLRP6 siRNA or overexpression lentivirus, co-cultured with primary neurons, following OGD(4 h)/R(24 h). (A) CCK8 assay was used to detect survival of neurons. (B, C) Flow cytometry assay was used to analyze cell apoptosis. Data are presented as means ± SEM. # P < 0.05, ## P < 0.01, versus NC group.
CRediT authorship contribution statement
reduce the level of GSDMD-NT and decrease the neurons pyroptosis. Briefly, we firstly demonstrated that NLRP6 induced pyroptosis of neurons by activating caspase-1 after OGD/R. In this study, our experiments showed that the impact of NLRP6 following OGD/R models in nerve cells, and suggested a novel treatment scheme for cerebral I/R injury.
Jinyan Zhang: Conceptualization, Methodology, Investigation, Writing - original draft. Ning Jiang: Software, Formal analysis, Investigation, Validation. Luyu Zhang: Resources, Data curation. Changchang Meng: Software, Methodology, Visualization. Jing Zhao: Conceptualization, Supervision, Project administration, Funding acquisition. Jingxian Wu: Conceptualization, Writing - review & editing, Project administration, Funding acquisition.
5. Conclusions Taken together, we found that NLRP6 expression increased predominantly in astrocytes subjected to OGD/R. In summary, the results identified that NLRP6 regulated inflammation and induced pyroptosis via the form of inflammasomes after OGD/R.
Acknowledgments This work was supported by grants from the National Natural Science Foundation of China, China (No. 81671158 and 81771261), National Youth Foundation of China, China (No. 81801183). Natural Science Foundation of Chongqing Science and Technology Committee, 6
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Fig. 4. NLRP6 binds to ASC, then activating caspase-1 to induce the release of inflammatory factors. (A) The cultured primary astrocytes were transfected with NLRP6 siRNA or overexpression lentivirus, following OGD(4 h)/R(24 h), proteins were immunoprecipitated with an anti-ASC or anti-NLRP6 antibody and measured by western blot analysis. (B) The cultured primary astrocytes were transfected with NLRP6 overexpression lentivirus, Ac-YVAD-cMK was simultaneously added to the LV-NLRP6- Ac-YVAD-cMK group, following OGD(4 h)/R(24 h), expression of NLRP6, pro–caspase-1, cleaved–caspase-1, IL-1β and IL-18 were measured by Western blot. Relative quantification of NLRP6 (C), cleaved–caspase-1(D), IL-1β(E) and IL-18(F). Data are presented as means ± SEM. # P < 0.05, versus NC group; & P < 0. 05, && P < 0. 01, versus OGD/R + LV-NLRP6 group.
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Fig. 5. NLRP6 mediates pyroptosis by activating Caspase-1 in neurons co-cultured with astrocytes after OGD/R. (A) The primary neurons co-cultured with astrocytes were transfected with NLRP6 siRNA or overexpression lentivirus, following OGD(4 h)/R (24 h), expressions of NLRP6 and GSDMD-NT were measured by Western blot analysis. (B) The primary neurons co-cultured with astrocytes were transfected with NLRP6 overexpression lentivirus, and Ac-YVAD-cMK was simultaneously added to the LV-NLRP6-Ac-YVAD-cMK group, following OGD(4 h)/R(24 h), expression of cleaved–caspase-1 and GSDMD-NT were measured by Western blot analysis. (C, D) Relative quantification of NLRP6 and GSDMD-NT of (A). (E, F) Relative quantification of cleaved–caspase-1 and GSDMD-NT of (B). (G, H) Cells were stained with PI and caspase1–FITC for flow cytometric analysis. Data are presented as means ± SEM. # P < 0.05, ## P < 0.01, versus OGD or NC group.; & P < 0. 05, && P < 0. 01, versus OGD + LV-NLRP6 group.
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NLRP6 expressed in astrocytes aggravates neurons injury after OGD/R through activating the inflammasome and inducing pyroptosis ⁎
Jinyan Zhanga,1, Ning Jianga,1, Luyu Zhangb, Changchang Mengc, Jing Zhaoc,d, , Jingxian Wua,d,
T ⁎
a
Department of Pathology, Chongqing Medical University, Chongqing, People’s Republic of China Department of Molecular Medicine, Chongqing Medical University, Chongqing, People’s Republic of China c Department of Pathophysiology, Chongqing Medical University, Chongqing, People’s Republic of China d Institute of Neuroscience, Chongqing Medical University, Chongqing, People’s Republic of China b
A R T I C LE I N FO
A B S T R A C T
Keywords: NLRP6 Stroke OGD/R Inflammation Pyroptosis Inflammasomes
NLRP6, the nucleotide oligomerization domain-like receptor family pyrin domain containing 6, has a substantiable effect on inflammation and host defense against microorganisms. In our previous study, NLRP6 promotes inflammation after cerebral I/R injury in a MCAO model. However, the effect of NLRP6 in different nerve cells subjected to OGD/R needs to be further understood. Here, evidence shows that the expression of NLRP6 is increased in different nerve cells subjected to OGD/R, and mainly expressed in astrocytes. NLRP6 may up-regulate inflammation factors (IL-1β, Il-8) via the form of inflammasomes in astrocytes after OGD/R. Then, primary neuron-astrocyte co-culture model under OGD/R in vitro was performed, and we found that NLRP6 decreased the neurons viability and aggravated apoptosis of neurons. Mechanically, NLRP6 could induce pyroptosis to regulate the survival of neurons through activating caspase-1.
1. Introduction Stroke is the second primary incentive of death of human as well as a crucial source of being disable. About 87% stroke patients are ischemic [1]. Recent evidence has indicated that inflammasomes are key intracellular receptors and sensors in innate immune system responsible for the initiation of sterile inflammation in CNS (central nervous system) diseases, for instance, traumatic brain injury [2], ischemic stroke [3], and ICH (intracerebral hemorrhage) [4]. Inflammasomes are crucial elements in stroke pathology [5] and necessary for inflammatory cytokine maturation [6]. NLRs (the nucleotide oligomerization domain-like receptor family) are currently known to assemble inflammasome complexes and to recognize pathogen associated molecular patterns (PAMPs). NLRs can also recruit pro-caspase-1 to form inflammasomes by binding to the adapter protein ASC (apoptosis-associated speck-like protein containing a caspase recruitment domain), being critical to caspase-1 initialization and subsequent secretion of IL-18 and IL-1β [7]. The previous study reveals that NLRP3 inflammasome hinderance played a neuroprotective role in cerebral I/R injury [8,9]. Besides, GSK-3β (glycogen synthase kinase 3β) inhibition-induced protection effect in cerebral I/R injury is partially attributed to the inhibition of NLRP3 inflammasomes [10]. In
addition, ever increasing evidence has uncovered that inflammasomemediated pyroptosis is engaged in the pathological progression of I/R injury in cerebral tissues. Caspase-1 is actuated on inflammasomes. Caspase-1 activation promotes pro- IL-1β and pro-IL-18 in activation, which is considered as the rate limiting step in inflammation. Caspase-1 activation also cleaves the gasdermins (GSMDs), which is responsible for the pore formation in cell membrane and subsequent pyroptosis [11,12]. Recently one study showed that blockage of NLRP3-induced pyroptosis contributed to the neuroprotective effects exerted by hispidulin via the AMPK/GSK-3β (AMP-activated protein kinase/glycogen synthase kinase 3β) signaling pathway [13]. From the above, inflammasome-mediated inflammation and pyroptosis play a key role in I/R injury of the brain. NLRP6, a member of the NLRs family, which is similar to other NLRs, could participate in inflammasome formation [14]. Our previous study firstly revealed that the level of NLRP6 was increased in a rat model of MCAO and inhibition of NLRP6 alleviated inflammation [15]. Additionally, it has been demonstrated that pyroptosis of gingival fibroblasts can be triggered by NLRP6 via activating caspase-1 [16]. However, it’s interesting to find that NLRP6 could be independent of the initialization of the inflammasome and have an important effect on protecting mice against colitis by reprogramming the gut microbiota
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Corresponding authors at: Department of Pathology, Chongqing Medical University, Chongqing, People’s Republic of China. E-mail addresses: [email protected] (J. Zhao), [email protected] (J. Wu). 1 These authors equally contributed to the work. https://doi.org/10.1016/j.intimp.2019.106183 Received 2 October 2019; Received in revised form 21 December 2019; Accepted 31 December 2019 1567-5769/ © 2020 Elsevier B.V. All rights reserved.
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and synthesized by GenePharma Corporation (Shanghai, China). In accordance with the NLRP6 gene of rat, the siRNA sequences (sense: 5′-GCAGACGAAUUGCCUACUUTT-3′; anti-sense: 5′-AAGUAGGCAAUU CGUCUGCTT-3′), and the control siRNA (sense: 5′-UUCUCCGAACGU GUCACGUTT-3′; antisense: 5′-ACGUGACACGUUCGGAGAATT-3′) were designed. Lipofectamine 2000 (Invitrogen) was used for siRNA transfection. Western blot was employed to examine the level of NLRP6.
[17]. Another study also showed that NLRP6 promoted the recovery of peripheral nerve injury by inhibiting the inflammatory response independent of inflammasomes [18]. In brief, these studies revealed that NLRP6 was associated with inflammation injury, which may or may not be dependent on inflammasomes. However, the function of NLRP6 in ischemia stroke is largely unknown, and its further mechanism still needs full investigation. Therefore, we hypothesized that NLRP6 may influence inflammation and pyroptosis after OGD/R in neural cells through inflammasome formation, and play an important role in the cerebral I/R injury.
2.5. Lentiviral transfection of NLRP6 overexpression NLRP6 overexpression lentivirus was purchased from GenePharma Corporation (Shanghai, China). No inserts in scramble lentivirus. Astrocytes were transfected with overexpression or scramble lentivirus in the medium to achieve a MOI (multiplicity of infection) of 15. GFP (green fluorescent protein)-positive cells were overexpressed as well as scramble cells. RFP (red fluorescent protein)-positive cells were astrocytes. The fluorescence microscope was used to confirm GFP and RFP cells. Western blot was employed to determine sustained NLRP6 overexpression efficiency.
2. Materials and methods 2.1. Cell culture We isolated astrocytes from Sprague-Dawley (SD) rats (aged one day), and neurons were isolated from SD rat embryos (gestational 17 days). Cell line of microglia (hapi) was donated by Institute of Neuroscience of Chongqing Medical University. Further details will be given afterwards.
2.6. q-PCR arrays 2.2. Co-culture Total RNA was isolated from cells using TRIzol reagent (Invitrogen) following manufacturer manual, and RNA concentration was measured by NanoDrop 8000 (Thermo Fisher Scientific). Reverse transcription was performed using a GoScript reverse transcription system (Promega) according to the manufacturer’s protocol. Then, q-PCR was performed using TaKaRa SYBR Premix Ex Taq II (Tli RNase H Plus) (TaKaRa Biotechnology) with a CFX96 Touch™ Real-Time PCR Detection System. Primer sequences were used for the amplification of NLRP6 (5′-CCGC ATCGTCTACTGTTCATCCTG-3′; 3′-GCACTCTCAAGCCACTCG-TAGC-5′) and GAPDH (5′-GACATGCCGCCTGGAGAAAC −3′; 3′-AGCC CAGGAT GCCCTTTAGT-5′). GAPDH was taken as an endogenous control.
Primary astrocytes were prepared by using of Park and colleagues [19] and plated in 75-cm2 T-flasks. After 2–3 weeks of culture, microglia and oligodendrocytes which growing above the astrocytes layer were detached from the flasks by mild shaking. The remaining adherent astrocytes were detached with trypsin and plated onto 14-mm poly-Llysine-coated glass coverslips in 24-well plates. When astrocytes were adhered on the glass coverslips in 24-well plates and almost reached 70–80% confluence, primary neurons were prepared by using previously published methods [20]. Primary neurons were plated directly on the plastic surface of 24-well plates coated with poly-L-lysine. Neuron purity (> 90%) was assessed using the neuron-specific marker, NeuN. Non-contact neuron-astrocyte co-cultures were established as previously described with modification [21]. The co-culture model is shown in the figure below. In brief, primary neurons were adhered on the plastic surface in 24-well platea, they were incubated with primary astrocytes which adhered on the glass coverslips on paraffin columns in the culture medium containing 2% B27 and 1% Pen/Strep. Here, paraffin columns which were not harmful to co-culture were used to separate neurons and astrocytes.
2.7. Western blot analysis A RIPA (radioimmunoprecipitation assay) lysis buffer (provided by Beyotime) accompanied with protease inhibitor (Roche, Basel, Switzerland) was utilized to separate total protein from the cultured cell. Protein electrophoresis was conducted on an 8–15% SDS/polyacrylamide gel (SDS/PAGE) and protein samples were then transferred to the PVDF membrane, which was then blocked by TBS-Tween buffer (pH 7.5; 5% nonfat milk, 20 mM Tris-HCL, 0.05% Tween-20, and 150 mM NaCl) for 2 h under room temperature. The membrane was subjected to incubation overnight at 4C. Following contents were used during incubation: primary antibodies for NALP6 (Thermo Fisher Scientific, USA); IL-18 (Protein tech, China); IL-1β (Affinity, USA); caspase-1 (Cell Signaling Technology, USA); ASC (Santa Cruz Biotechnology, USA), GSDMD-NT (Abcam, USA) and β-actin (Proteintech, China). TBS-T washing was performed three times with an interval of 10 min for the membrane, which was then incubated with the secondary antibody (anti-rabbit or anti-rat IgG) for 2 h under room temperature, followed by TBS-T washing for three times with an interval of 10 min. Enhanced chemiluminescence (ECL) reagent was added following the manufacturer manual (Bio-Rad). Afterwards, X-ray was used to detect the signals from the ECL.
2.3. OGD/R To stimulate ischemic-like circumstance outside in vitro, cell culture was subjected to OGD (oxygen-glucose deprivation) and subsequently transferred into the original medium with oxygen (95% air, 5% CO2) as well as glucose for 24 h. Anaerobic chamber (Thermo Scientific) with gas mixtures (1% O2, 5% CO2 as well as 94% N2) and deoxygenated glucose-free Hanks' Balanced Salt Solution (provided by Invitrogen) was used for OGD.
2.8. Immunofluorescence staining Astrocytes, microglia and neuronal cells were cultured on coverslips and exposed to OGD for 0, 1, 2, 4, or 6 h, respectively. Then the injured cultured cells were reperfused for twenty-four hours and fixed using paraformaldehyde (4%) in PBS. The coverslip was washed twice with PBS, followed by incubation with a primary antibody at four centigrade degree overnight. Then, the coverslip was incubated one hour with a fluorescein-labelled secondary antibody. DAPI was used to stain cellular
2.4. SiRNA transfection of NLRP6 Small interfering RNA (siRNA) sequences of NLRP6 were designed 2
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nuclei. Cells were imagined with an Olympus confocal fluorescence microscope.
3.2. The effect of NLPR6 intervention on inflammatory factors The results above showed that NLRP6 increased in astrocytes after OGD/R. And NLRP6 was related to inflammation, so this experiment intended to analyze the relationship between NLRP6 and inflammation in astrocytes after OGD/R. We then constructed siRNA of NLRP6 and NLRPR overexpression lentivirus to transfect astrocytes subjected to OGD/R. Western blot and ELISA were applied to measure levels of IL18, IL-1β, and Caspase-1. NLRP6 silence decreased the protein expression of the inflammatory factors IL-18, IL-1β, and Caspase-1, while NLRP6 overexpression induced a significant expression of IL-18, IL-1β, and Caspase-1 (Fig. 2A, B, C, D). Meanwhile, the data of ELISA were consistent with the preceding results (Fig. 2F and G). The results from this part of experiments revealed that NLRP6 may play a pro-inflammatory role in astrocytes after OGD/R.
2.9. Cell viability Cell Counting Kit-8 (CCK-8, Kumamoto, Japan) was implemented for neuronal viability assay following manufacturer manual. In brief, CCK-8 solutions were introduced to each coverslip containing OGD/R cells and the absorbance at 450 nm was examined by a microplate reader. 2.10. ELISA (enzyme-linked immunosorbent assay) Expression levels of IL-1B and IL-18 in the cell supernatants were detected using the relative rat ELISA Kit (Dakewe, China). 2.11. Co-immunoprecipitation
3.3. The effect of NLRP6 in neurons co-culture with astrocytes following OGD/R
Cultured primary astrocytes were harvested and total protein was isolated. Co-immunoprecipitation (Co-IP) was conducted following manufacturer's instruction. Anti-ASC (Santa Cruz Biotechnology, USA), anti-NLRP6 antibody (Bioss, China), and protein A/G agarose (Beyotime, China) were used. After incubation the cells with beads, they underwent centrifugation at 2,500g for 5 min, a total of 5 times. Co-IP was examined by Western blotting.
To determine the role of NLRP6 in primary neurons with primary astrocytes co-cultured in 24-well plates, we used NLRP6 siRNA and NLRP6 overexpressing lentivirus following OGD/R, and then the viability and apoptosis of neurons were detected by CCK8 assay and FCM (flow cytometry). Results showed that NLRP6 knockdown significantly caused a reduced percentage of neuronal apoptosis and promoted neuronal viability, whereas NLRP6 overexpression emerged the opposite effects (Fig. 3A, B, C). These data indicated that NLRP6 may exert a non-protective function in neurons-astrocytes co-culture model after OGD/R injury.
2.12. Flow cytometry After the cells subjected to OGD/R, removing medium, and wash the cells twice with PBS slowly. Removing the PBS, adding 1 mL of 0.125% trypsin in every well and placing the plate to stand at 37 °C incubator for 20 min, the plate with mild shaking every 5 min to accelerate cell shedding. Then, adding 1 mL neuronal culture medium to stop digestion. Pipetting the cells off the plate, and transferring them to centrifuge tube. Pelleting the cells by centrifugation at 1000g for 5 min. Removing the medium and resuspending the cells in PBS. Counting and adjusting the cells to 1 × 106 cells/mL in PBS. Cells are labeled by PI (Propidium Iodide) and FITC-coupled annexin V (annexin V-FITC) double staining for detection of neurons apoptosis. To specifically block caspase-1, AcYVAD-cmk (APExBIO, USA) was used. Caspase-1-FITC (Immunochemistry Technologies, USA) was used to examine the activity of caspase-1. The apoptosis and pyroptosis were identified by flow cytometry (CytoFLEX, USA).
3.4. NLRP6 binds to ASC, then activating caspase-1 to induce the release of inflammatory factors To identify whether NLRP6 activates the inflammasome pathway, co-immunoprecipitation assay was conducted. NLRP6-ASC was more effective in co-precipitating of astrocytes infected via NLRP6 overexpression lentivirus while NLRP6-ASC was less co-precipitated in astrocytes infected by NLRP6 siRNA (Fig. 4A). Lentiviral overexpression of NLRP6 promoted pro-caspase-1 cleavage into activation form in astrocytes, which was inhibited by Ac-YVAD-cmk (caspase-1 inhibitor) (Fig. 4B). The effects of NLRP6 overexpression on the secretion of IL-18 and IL-1β were detected by Western blotting analysis. The analysis exhibit that lentiviral overexpression of NLRP6 elevated the protein expression levels of IL-18 and IL-1β, which was blocked by caspase-1 inhibitor Ac-YVAD-cmk (Fig. 4B). These findings suggested that the increase of IL-18 and IL-1β secretion was attributed to NLRP6-induced caspase-1 activation in astrocytes.
2.13. Statistical analysis Values were described as mean ± SEM, and one-way ANOVA, twoway ANOVA or t test with GraphPad Prism 7.0 software was employed for statistical analysis. P < 0.05 meant significant difference. 3. Results
3.5. NLRP6 mediates pyroptosis by activating Caspase-1 in neurons coculture with astrocytes after OGD/R
3.1. The expression of NLRP6 in neurons, astrocytes and microglia cells To further discuss the relationship between NLRP6 and pyroptosis after OGD/R models, we firstly detected the level of GSDMD-NT with NLRP6 knockdown or overexpression in neurons-astrocytes co-cultured model after OGD 4 h. Then, we identified whether NLRP6 induced pyroptosis through activating Caspase-1. NLRP6 silence decreased the protein levels of the pyroptosis related factors GSDMD-NT, while NLRP6 overexpression induced a significant expression of GSDMD-NT (Fig. 5A, C, D). In addition, Ac-YVAD-CMK (Caspase-1 inhibitor) can rescue NLRP6-mediated pyroptosis in co-cultured cells after OGD/R (OGD/R + LV-NLRP6 group vs OGD/R + LV-NLRP6 + Ac-YVAD-CMK group, Fig. 5B, E, F), indicating that NLRP6 mediated pyroptosis by activating Caspase-1. As illustrated in Fig. 5G and H, the trend of FCM is consistent with the results of Western blot.
To investigate the effect of NLRP6 in OGD/R models, we first used immunofluorescence to evaluate the expression of NLRP6 in primary neurons, astrocytes and microglia cells. As labelled by immunofluorescence staining for NLRP6 and neuronal marker NeuN, astrocytic marker GFAP, and microglial maker Iba-1, and we found that NLRP6 was mainly expressed in astrocytes and reached the peak at 4 h after OGD/R (Fig. 1B), but little expressed in neurons and microglia (Fig. 1A and C). Then, western blot was utilized to investigate the expression of NLRP6 in cultured primary astrocytes after OGD/R and found that NLRP6 protein was markedly increased in the OGD/R, with the peak expression of NLRP6 at 4 h after OGD/R (Fig. 1D and E). The mRNA level of NLRP6 was consistent with results of western blots (Fig. 1F). 3
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Fig. 1. The expression of NLRP6 in neuron, astrocyte and microglia cells. (A) Immunofluorescence staining for NeuN and NLRP6; (B) Immunofluorescence staining for GFAP and NLRP6; (C) Immunofluorescence staining for IBA1 and NLRP6; (D) Western blot of NLRP6 expression in sham, 1, 2, 4, 6 h after OGD in astrocytes; (E) Relative quantification of NLRP6. (F) The mRNA level of NLRP6 in sham, 1, 2, 4, 6 h after OGD in astrocytes. Data are presented as means ± SEM, * P < 0.05, ** P < 0.01, *** P < 0.001 versus sham group. NeuN: neuron-specific nuclear protein; GFAP: glial fibrillary acidic protein; Iba-1: ionized calcium binding adapter molecule 1.
4. Discussion
whether it is dependent on the inflammasome-induced by NLRP6. In our previous study, we found that NLRP6 promoted the release of inflammation factors (IL-1β, IL-8) after I/R injury in the brain tissues of MCAO models [15]. Our investigation showed that NLRP6 also upregulated inflammation factors (IL-1β, IL-18) in astrocytes after OGD/R models, which is consistent with our previous findings. In addition, our experimental data demonstrated that NLRP6 interacted with ASC, and inhibiting caspase-1 could decrease the release of inflammatory factors. These results indicated that similar to other NLRs, NLRP6 played a proinflammation effect in OGD/R models in astrocytes dependent on inflammasomes. In recent years, many researches focus on an inflammatory cell death mode-pyroptosis. Pyroptosis is an inflammatory form of programmed cell death, which is characterized by activation of caspase-1 followed by IL-18 and IL-1β, and cellular extravasation [28]. Within the canonical pathway, pyroptosis is characterized by initialization of inflammasomes, and then activated caspases-1 cleaves GSDMD to generate GSDMD-NT, which further induces membrane pore formation and mediates proptosis [29]. Pyroptosis has been identified to be activated both in vivo and in vitro stroke models [30]. More importantly, pyroptotic neuronal and glial cell death trigged by stroke, contributes to neurological deficits, neuroinflammation and the imbalance of homeostasis in the CNS [31]. A recent report shows that pyroptosis of gingival fibroblasts could be attributed to NLRP6 induced activation of caspase-1 [16]. In our preliminary experiment, we found that NLRP6 may influence the survival of neurons, because the increased expression of NLRP6 significantly decreased the neurons vitality and aggravated neurons apoptosis. In the meantime, we identified that NLRP6 could trigger GSDMD-NT in primary neurons-astrocytes co-cultured model after OGD/R. Furthermore, we added Ac-YVAD-CMK, a peptide inhibitor of caspase-1, and it was found that inhibition of caspase-1 could
For the first time, we found that OGD/R elevated the protein expression level of NLRP6 and NLRP6 was largely colocalized with GFAP in the primary cultured astrocytes. We also illustrated that NLRP6 enhanced the release of inflammatory factors via the form of inflammasomes. In addition, intervention of NLRP6 may influence the viability and apoptosis of neurons. More importantly, it is the originality that NLRP6 regulated pyroptosis by activating Caspase-1 in neurons-astrocytes co-cultured model after OGD/R. Previous reports have indicated that NLRP6 is expressed in different tissues, including the kidney, liver, lung, stomach, pancreas, spleen and small and large intestines. Due to the relative high expression of NLRP6 in the intestines, studies of NLRP6 function largely focus on the intestines [22–24]. Recently, increasing evidence suggests that NLRs critically affect ischemic stroke pathogenesis, development and progression [7]. Moreover, blockage of NLRP3 inflammasomes at different cellular levels may provide some therapeutic strategies against neurological deterioration in ischemic stroke [25]. While it remains unclear about the effects of I/R injury in brain tissues on the expression and function of NLRP6 in the CNS. For the first time, our results showed that NLRP6 was principally expressed in the astrocytes and the resulting expression was increased after OGD/R. The pathophysiology of cerebral I/R injury is complex, which has multiple damage and cascade reactions, including inflammation, oxidative stress and cell death [26]. NLRP6, called PYPAF5 initialy, has been implicated to activate caspase1 in the inflammasome signaling, which correspondently activates IL1β and IL-18 [27]. However, emerging studies show that NLRP6 protects against colitis and colitis-related colorectal cancer via inflammasome-independent mechanisms [17]. Thus, it is very interesting to investigate the impact of NLRP6 on the I/R injury in the brain tissues, and 4
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Fig. 2. The effect of NLPR6 intervention on inflammation factors. The cultured primary astrocytes were transfected with NLRP6 interference lentivirus or overexpression lentivirus, following OGD(4 h)/R(24 h). (A) Western blot was used to measure the protein levels of NLRP6, cleaved–caspase-1, IL-1β and IL-18. Relative quantification of NLRP6 (B), cleaved–caspase-1 (C), IL-1β(D) and IL-18 (E). (F, G) ELISA assay was used to measure IL-1β and IL-18 levels in cell supernatants. Data are presented as means ± SEM. ** P < 0.01, versus sham group; # P < 0.05, ## P < 0.01, versus NC group.
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Fig. 3. The effect of NLRP6 in neurons co-cultured with astrocytes following OGD/R. The cultured primary astrocytes were transfected with NLRP6 siRNA or overexpression lentivirus, co-cultured with primary neurons, following OGD(4 h)/R(24 h). (A) CCK8 assay was used to detect survival of neurons. (B, C) Flow cytometry assay was used to analyze cell apoptosis. Data are presented as means ± SEM. # P < 0.05, ## P < 0.01, versus NC group.
CRediT authorship contribution statement
reduce the level of GSDMD-NT and decrease the neurons pyroptosis. Briefly, we firstly demonstrated that NLRP6 induced pyroptosis of neurons by activating caspase-1 after OGD/R. In this study, our experiments showed that the impact of NLRP6 following OGD/R models in nerve cells, and suggested a novel treatment scheme for cerebral I/R injury.
Jinyan Zhang: Conceptualization, Methodology, Investigation, Writing - original draft. Ning Jiang: Software, Formal analysis, Investigation, Validation. Luyu Zhang: Resources, Data curation. Changchang Meng: Software, Methodology, Visualization. Jing Zhao: Conceptualization, Supervision, Project administration, Funding acquisition. Jingxian Wu: Conceptualization, Writing - review & editing, Project administration, Funding acquisition.
5. Conclusions Taken together, we found that NLRP6 expression increased predominantly in astrocytes subjected to OGD/R. In summary, the results identified that NLRP6 regulated inflammation and induced pyroptosis via the form of inflammasomes after OGD/R.
Acknowledgments This work was supported by grants from the National Natural Science Foundation of China, China (No. 81671158 and 81771261), National Youth Foundation of China, China (No. 81801183). Natural Science Foundation of Chongqing Science and Technology Committee, 6
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Fig. 4. NLRP6 binds to ASC, then activating caspase-1 to induce the release of inflammatory factors. (A) The cultured primary astrocytes were transfected with NLRP6 siRNA or overexpression lentivirus, following OGD(4 h)/R(24 h), proteins were immunoprecipitated with an anti-ASC or anti-NLRP6 antibody and measured by western blot analysis. (B) The cultured primary astrocytes were transfected with NLRP6 overexpression lentivirus, Ac-YVAD-cMK was simultaneously added to the LV-NLRP6- Ac-YVAD-cMK group, following OGD(4 h)/R(24 h), expression of NLRP6, pro–caspase-1, cleaved–caspase-1, IL-1β and IL-18 were measured by Western blot. Relative quantification of NLRP6 (C), cleaved–caspase-1(D), IL-1β(E) and IL-18(F). Data are presented as means ± SEM. # P < 0.05, versus NC group; & P < 0. 05, && P < 0. 01, versus OGD/R + LV-NLRP6 group.
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Fig. 5. NLRP6 mediates pyroptosis by activating Caspase-1 in neurons co-cultured with astrocytes after OGD/R. (A) The primary neurons co-cultured with astrocytes were transfected with NLRP6 siRNA or overexpression lentivirus, following OGD(4 h)/R (24 h), expressions of NLRP6 and GSDMD-NT were measured by Western blot analysis. (B) The primary neurons co-cultured with astrocytes were transfected with NLRP6 overexpression lentivirus, and Ac-YVAD-cMK was simultaneously added to the LV-NLRP6-Ac-YVAD-cMK group, following OGD(4 h)/R(24 h), expression of cleaved–caspase-1 and GSDMD-NT were measured by Western blot analysis. (C, D) Relative quantification of NLRP6 and GSDMD-NT of (A). (E, F) Relative quantification of cleaved–caspase-1 and GSDMD-NT of (B). (G, H) Cells were stained with PI and caspase1–FITC for flow cytometric analysis. Data are presented as means ± SEM. # P < 0.05, ## P < 0.01, versus OGD or NC group.; & P < 0. 05, && P < 0. 01, versus OGD + LV-NLRP6 group.
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Chongqing, China (No. cstc2015jcyjBX0144). All the authors declare that there is no conflict of interest for any of them.
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