MSC-derived exosomal miR-140-3p improves cognitive dysfunction in sepsis-associated encephalopathy by HMGB1 and S-lactoylglutathione metabolism

Abstract

MiRNAs in mesenchymal stem cells (MSCs)-derived exosome (MSCs-exo) play an important role in the treatment of sepsis. We explored the mechanism through which MSCs-exo influences cognitive impairment in sepsis-associated encephalopathy (SAE). Here, we show that miR-140-3p targeted Hmgb1. MSCs-exo plus miR-140-3p mimic (Exo) and antibiotic imipenem/cilastatin (ABX) improve survival, weight, and cognitive impairment in cecal ligation and puncture (CLP) mice. Exo and ABX inhibit high mobility group box 1 (HMGB1), IBA-1, interleukin (IL)-1β, IL-6, iNOS, TNF-α, p65/p-p65, NLRP3, Caspase 1, and GSDMD-N levels. In addition, Exo upregulates S-lactoylglutathione levels in the hippocampus of CLP mice. Our data further demonstrates that Exo and S-lactoylglutathione increase GSH levels in LPS-induced HMC3 cells and decrease LD and GLO2 levels, inhibiting inflammatory responses and pyroptosis. These findings suggest that MSCs-exo-mediated delivery of miR-140-3p ameliorates cognitive impairment in mice with SAE by HMGB1 and S-lactoylglutathione metabolism, providing potential therapeutic targets for the clinical treatment of SAE.

Fig. 1. MiRNAs targeting HMGB1 in SAE.

a Volcano map showing DEmiRNAs in the control group and SAE group. b Heatmaps showing the variations in miRNA abundance. c Venn diagram showing the intersection of DEmiRNAs and HMGB1-targeting miRNAs.

Fig. 2. Verification of the expression of HMGB1-targeting miRNAs in MSCs-exo.

a Morphological observation of MSCs. Scale bars: ×100 images, 100 µm; ×200 images, 50 µm. b Flow cytometric analysis of MSC surface markers. c Bone and lipid-induced differentiation of MSCs was observed by alizarin red staining and oil red O staining, respectively. Scale bars: ×100 images, 100 µm; ×400 images, 25 µm. d TEM and NTA were used to observe the morphology of exosomes derived from MSCs. Scale bars: 200 nm. e Western blot analysis of exosome surface markers (TSG101, HSP70, and CD63). f The expression of the top 10 miRNAs in MSCs-exo was measured by qRT-PCR. Each experiment was independently repeated three times. g The box diagram showing the miR-140-3p level in the control (n = 3) and SAE (n = 6) groups.

Fig. 3. MSCs-exo-mediated delivery of miR-140-3p inhibited LPS-induced HMC3 cell inflammation and pyroptosis.

a MiR-140-3p levels were measured by qRT-PCR. b IBA-1 and HMGB1 levels in HMC3 cells were examined by immunofluorescence analysis. Scale bars: 25 µm. c, d ELISA was used to examine iNOS, IL-1β, IL-6, TNF-α, and HMGB1 levels in the supernatant of HMC3 cells. e Western blot analysis of p65, p-p65, NLRP3, and HMGB1 expression. f Bioinformatics analysis was used to predict the binding sites of miR-140-3p to HMGB1 (human). g The dual-luciferase reporter assay confirmed the binding of miR-140-3p to HMGB1. h The uptake of MSCs-exo was observed by immunofluorescence analysis. Scale bars: 25 µm. Each experiment was independently repeated three times. Error bars show SD. *P < 0.05 vs. the normal group; #P < 0.05 vs. the LPS group; &P < 0.05 vs. the MSCs-exo group.

Fig. 4. MSCs-exo-mediated delivery of miR-140-3p improved cognitive impairment in mice with SAE.

a Bioinformatics analysis was used to predict the binding sites of miR-140-3p to Hmgb1 (mouse). b The dual-luciferase reporter assay confirmed the binding of miR-140-3p to Hmgb1. c Survival rate analysis of CLP mice. d Changes in the weight of CLP mice. e Morris water maze test. n = 10. Error bars show SD. *P < 0.05 vs. the sham group; #P < 0.05 vs. the model group; & P < 0.05 vs. the ABX group.

Fig. 5. MSCs-exo-mediated delivery of miR-140-3p reduced neuroinflammation in mice with SAE.

a, b Hippocampal apoptosis was observed by TUNEL staining. Scale bars: 25 µm. c, d IBA-1 and HMGB1 levels in hippocampal tissues were examined by immunohistochemistry. Scale bars: 25 µm. e ELISA was used to measure IL-1β, IL-6, and TNF-α levels in the hippocampus. n = 3. Error bars show SD. *P < 0.05 vs. the sham group; #P < 0.05 vs. the model group; & P < 0.05 vs. the Exo group.

Fig. 6. MSCs-exo-mediated delivery of miR-140-3p reduced pyroptosis in mice with SAE.

a Western blot analysis of HMGB1, p65, p-p65, and NLRP3 expression in the hippocampus. b, c NLRP3 and Caspase 1 levels in the hippocampus were examined by immunohistochemistry. Scale bars: ×100 images, 100 µm; ×400 images, 25 µm. d Western blot analysis of Caspase 1 and GSDMD levels in the hippocampus. n = 3. Error bars show SD. *P < 0.05 vs. the sham group; #P < 0.05 vs. the model group; & P < 0.05 vs. the Exo group.

Fig. 7. MiR-140-3p delivered by MSCs-exo changed the metabolic profile of the hippocampus of mice with SAE.

a Principal component analysis. b Volcano plot showing differentially expressed metabolites in the groups. c Heatmap showing differences in metabolites between the groups. d Box plots showing key differentially expressed metabolites. Error bars show SD. *P < 0.05 vs. the sham group; #P < 0.05 vs. the model group.

Fig. 8. Overview of enriched metabolite sets between different groups.

Effects of miR-140-3p delivered by MSCs-exo on metabolic function in the hippocampus of mice with SAE.

Fig. 9. S-lactoylglutathione supplementation in combination with MSCs-exo delivery of miR-140-3p inhibited LPS-induced HMC3 cell inflammation.

a GSH and LD levels were measured by ELISA. b IBA-1 and HMGB1 levels in HMC3 cells were examined by immunofluorescence analysis. Scale bars: 25 µm. c ELISA was used to measure iNOS, IL-1β, IL-6, TNF-α, and HMGB1 levels in the supernatant of HMC3 cells supernatant. Each experiment was independently repeated three times. Error bars show SD. *P < 0.05 vs. the LPS group; #P < 0.05 vs. the Exo group.

Fig. 10. S-lactoylglutathione supplementation in combination with MSCs-exo delivery of miR-140-3p inhibited LPS-induced HMC3 cell pyroptosis.

a and b NLRP3 and Caspase 1 expression were examined by immunofluorescence analysis. Scale bars: 25 µm. c Western blot analysis of HMGB1, GLO2, p65, p-p65, NLRP3, Caspase 1, and GSDMD levels. Each experiment was independently repeated three times. Error bars show SD. *P < 0.05 vs. the LPS group; #P < 0.05 vs. the Exo group.