Astrocyte-derived exosomal miR-378a-5p mitigates cerebral ischemic neuroinflammation by modulating NLRP3-mediated pyroptosis

Abstract

Objective: This study aimed to investigate the regulatory role of astrocyte-derived exosomes and their microRNAs (miRNAs) in modulating neuronal pyroptosis during cerebral ischemia.

Methods: Astrocyte-derived exosomes were studied for treating cerebral ischemia in both in vitro and in vivo models. The effects of astrocyte-derived exosomes on neuroinflammation were investigated by analyzing exosome uptake, nerve damage, and pyroptosis protein expression. High throughput sequencing was used to identify astrocyte-derived exosomal miRNAs linked to pyroptosis, followed by validation via qRT‒PCR. The relationship between these miRNAs and NLRP3 was studied using a dual luciferase reporter assay. This study used miR-378a-5p overexpression and knockdown to manipulate OGD injury in nerve cells. The impact of astrocyte-derived exosomal miR-378a-5p on the regulation of cerebral ischemic neuroinflammation was assessed through analysis of nerve injury and pyroptosis protein expression.

Results: Our findings demonstrated that astrocyte-derived exosomes were internalized by neurons both in vitro and in vivo. Additionally, Astrocyte-derived exosomes displayed a neuroprotective effect against OGD-induced neuronal injury and brain injury in the ischemic cortical region of middle cerebral artery occlusion (MCAO) rats while also reducing pyroptosis. Further investigations revealed the involvement of astrocyte-derived exosomal miR-378a-5p in regulating pyroptosis by inhibiting NLRP3. The overexpression of miR-378a-5p mitigated neuronal damage, whereas the knockdown of miR-378a-5p increased NLRP3 expression and exacerbated pyroptosis, thus reversing this neuroprotective effect.

Conclusion: Astrocyte-derived exosomal miR-378a-5p has a neuroprotective effect on cerebral ischemia by suppressing neuroinflammation associated with NLRP3-mediated pyroptosis.Further research is required to comprehensively elucidate the signaling pathways by which astrocyte-derived exosomal miR-378a-5p modulates neuronal pyroptosis.

Keywords: NLRP3; astrocyte-derived exosomes; cerebral ischemia; inflammation; miR-378a-5p; pyroptosis.

Figure 1

Identification of primary astrocytes, neurons, and astrocyte-derived exosomes was performed as follows. (A) Immunofluorescence assay of astrocytes revealed the presence of the marker GFAP. Scale bar = 20 µm. (B) Immunofluorescence staining was employed to detect the presence of the neuron marker NSE. Scale bar = 20 µm. (C) TEM was used to visualize exosomes produced from astrocytes. Scale bar = 100 nm. (D) The particle size distribution of astro-cyte-derived exosomes was evaluated using NTA. (E) Exosome marker protein expression levels were determined by Western blot analysis utilizing CD81 and TSG101.

Figure 2

Astrocyte-derived exosomes were internalized by OGD neurons and conferred protection against OGD-induced neuronal injury. (A, B) Cell viability of neurons was assessed using CCK8 in the presence or absence of astrocyte-derived exosomes. (C, D) LDH release rate was measured in neurons with or without astrocyte-derived exosomes. (E-G) Immunofluorescence staining was used to assess the uptake of exosomes from astrocytes and the manifestation of neuronal pyroptosis protein GSDMD. Scale bar = 25 µm. (H, I) GSDMD expression of the neuronal pyroptosis protein was measured using the Western-Blot method. The statistical data for every group is presented as the mean ± SD (n≥ 3). *P < 0.05, ** P < 0.01.

Figure 3

Astrocyte-derived exosomes attenuate OGD-induced neuronal pyroptosis. (A) Morphological observation of neurons. Scale bar = 100 µm. (B) The cell viability of neurons was assessed using CCK8. (C) LDH release rate of neurons. (D, E) GSDMD expression of the neuronal pyroptosis protein was measured using the Western-Blot method. The statistical data for every group is presented as the mean ± SD (n ≥ 3). *P < 0.05, ** P < 0.01.

Figure 4

Astrocyte-derived exosomes enhanced brain injury recovery and attenuated pyroptosis in rats subjected to MCAO. (A, B) Fluorescence imaging revealed the entry of PKH26-labeled astrocyte-derived exosomes into the brain. Scale bar = 25 µm. (C, D) Nissl staining was performed to assess neuronal survival. (E, F) Immunofluorescence staining demonstrated the expression of GSDMD, a key protein involved in pyroptosis, in cortical neurons on the ischemic side of rats. Scale bar = 50 µm. (G-J) The amounts of GSDMD, Caspase-1 p20, and NRP3 proteins in the cortical region on the ischemic side were assessed by Western blot analysis. The statistical data for every group is presented as the mean ± SD (n ≥ 3). *P < 0.05, ** P < 0.01.

Figure 5

Astrocyte-derived exosomal miR-378a-5p contributes to neuroprotection by targeting NLRP3. (A) The miRNAs from astrocyte-derived exosomes were compared with NLRP3-related miRNAs in the miRWalk database. (B-I) The miRNA expression was assessed using qRT-PCR. (J, K) Dual-luciferase assay was performed to confirm the specific binding site between miR-378a-5p and NLRP3 3'UTR. The statistical data for every group is presented as the mean ± SD (n≥ 3). *P < 0.05, ** P < 0.01.

Figure 6

Astrocyte-derived exosomal miR-378-5p attenuates neuronal pyroptosis after cerebral ischemia by down-regulating NLRP3. (A) Morphological observation of neurons. Scale bar = 100 µm. (B) CCK8 assay for neuronal cell viability. (C) LDH release rate of neurons. (D, E) NLRP3 expression of neurons detected by immunofluorescence. Scale bar = 20 µm. (F-I) Western-Blot assay for neuronal pyroptosis proteins NLRP3, Caspase-1p20 and GSDMD. The statistical data for every group is presented as the mean ± SD (n > 3). *P < 0.05, ** P < 0.01.

Figure 7

Schematic illustration of astrocyte-derived exosomal miR-378a-5p mitigates cerebral ischemic neuroinflammation by modulating NLRP3-mediated pyroptosis.