Exosomes derived from human umbilical cord mesenchymal stem cells decrease neuroinflammation and facilitate the restoration of nerve function in rats suffering from intracerebral hemorrhage

Abstract

Exosomes derived from human umbilical cord mesenchymal stem cells (hUCMSC-ex) have become a hopeful substitute for whole-cell therapy due to their minimal immunogenicity and tumorigenicity. The present study aimed to investigate the hypothesis that hUCMSC-ex can alleviate excessive inflammation resulting from intracerebral hemorrhage (ICH) and facilitate the rehabilitation of the nervous system in rats. In vivo, hemorrhagic stroke was induced by injecting collagenase IV into the striatum of rats using stereotactic techniques. hUCMSC-ex were injected via the tail vein at 6 h after ICH model establishment at a dosage of 200 µg. In vitro, astrocytes were pretreated with hUCMSC-ex and then stimulated with hemin (20 μmol/mL) to establish an ICH cell model. The expression of TLR4/NF-κB signaling pathway proteins and inflammatory factors, including TNF-α, IL-1β, and IL-10, was assessed both in vivo and in vitro to investigate the impact of hUCMSC-ex on inflammation. The neurological function of the ICH rats was evaluated using the corner turn test, forelimb placement test, Longa score, and Bederson score on the 1st, 3rd, and 5th day. Additionally, RT-PCR was employed to examine the mRNA expression of TLR4 following hUCMSC-ex treatment. The findings demonstrated that hUCMSC-ex downregulated the protein expression of TLR4, NF-κB/P65, and p-P65, reduced the levels of pro-inflammatory cytokines TNF-α and IL-1β, and increased the expression of the anti-inflammatory cytokine IL-10. Ultimately, the administration of hUCMSC-ex improved the behavioral performance of the ICH rats. However, the results of PT-PCR indicated that hUCMSC-ex did not affect the expression of TLR4 mRNA induced by ICH, suggesting that hUCMSCs-ex may inhibit TLR4 translation rather than transcription, thereby suppressing the TLR4/NF-κB signaling pathway. We can conclude that hUCMSC-ex mitigates hyperinflammation following ICH by inhibiting the TLR4/NF-κB signaling pathway. This study provides preclinical evidence for the potential future application of hUCMSC-ex in the treatment of cerebral injury.

Keywords: Exosomes; Human umbilical cord mesenchymal stem cells; Inflammation; Intracerebral hemorrhage; TLR4/NF-κB.

Fig. 1

Characterization of hUCMSCs and Exosomes. A Flow cytometry analysis showed hUCMSCs express CD73, CD90, and CD105 but not CD34 and CD45. B TEM analysis of hUCMSC-ex. Scale bar = 50 nm/100 nm. C Dynamic light scattering analysis of hUCMSC-ex. D Western blot identification results

Fig. 2

hUCMSC-ex inhibit ICH-induced inflammation and improve MMP in primary astrocytes. A Morphological characteristics of Primary astrocytes and their specific marker GFAP. B hUCMSC-ex transfected into primary astrocytes. C–E ELISA analysis for IL-1β TNF-α, and IL-10 levels in astrocytes supernatants. F Western blot for TLR4 and NF-κB/P65, p-P65. G Statistical analysis of TLR4. H Statistical analysis of TLR4 mRNA. (I) Representative images of the JC-1 staining. J The histogram of the ratio of JC-1 fluorescence (Red / Green) in the primary culture of astrocytes. (n = 3). Scale bars: A and H, 50 μm; B, 100 μm. Data were expressed as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 3

hUCMSC-ex reduce the brain injury caused by ICH. A Representative pictures of the hemorrhagic lesion. B Nissl staining in the Rat of different groups. C Representative pictures of the Evens blue extravasation of different groups. (n = 3). D The cerebral water content of each group was examined to evaluate the effect of treatment on cerebral edema. (n = 5). E The forelimb placement test, (n = 10). F The corner turn test. (n = 10). The G Longa and H Pedersen scores were performed to evaluate from day 1 to day 5 after ICH to assess recovery of neural function. (n = 10). Data were expressed as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 4

hUCMSC-ex alleviated the inflammatory response of rats after ICH. A Total WBC in different groups. B–D The concentrations of IL-1β, TNF-α, and IL-10 in serum were detected by ELISA. E Western blot for TLR4 and NF-κB/P65, p-P65. F Statistical analysis of TLR4. G Statistical analysis of TLR4 mRNA. H Representative immunohistochemical staining of MPO and CD68 in different groups and I and J statistical analysis (n = 3). Scale bars: 200 μm (100 ×), 50 μm (400 ×). Data were expressed as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001

Fig. 5

hUCMSC-ex inhibit ICH-induced hyperinflammation by targeting TLR4/NF-κB signaling pathway. A Western blot for TLR4 and NF-κB/P65, p-P65. B Statistical analysis of TLR4. C Statistical analysis of TLR4 mRNA. D–F ELISA analysis for IL-1β TNF-α, and IL-10 levels in different groups. G Representative immunofluorescence images of TLR4 expression in astrocytes and I statistical analysis. H Representative images of the nuclear translocation of P65 (white arrows) in ICH-induced astrocytes and J statistical analysis. (n = 3). Scale bars: G 50 μm; H 100 μm. Data were expressed as mean ± standard deviation. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001