Human UC-MSCs were purchased from ATCC (American Type Culture Collection, Manassas, VA, USA). UC-MSCs were cultured with Mesenchymal Stem Cell Basal Media containing growth factors (Mesenchymal Stem Cell Growth Kit, American Type Culture Collection, Manassas, VA, USA).

Eighteen male C57BL/6 mice aged 4–6 weeks were acquired from Beijing Laboratory Animal Research Center (Beijing, China). All mice were divided into three groups (6 mice in each group): sham group, LPS group, and LPS + UC-MSC group. To determine the effect of UC-MSCs on SAE, the SAE model was first established by injecting mice with lipopolysaccharide (LPS). Except for the mice in the sham group, 3 mg/kg LPS were injected into all mice intraperitoneally to induce SAE [4]. After 1 h post LPS injection, the mice in the LPS + UC-MSC group were injected with UC-MSCs (1 $\times$ 10${}^5$ cells) using a 35-G needle slowly administered over 1 min via the femoral vein. Eight days later, the cognitive function of mice was evaluated by the Morris water maze test. Mice were euthanized, and brain samples were collected after evaluation of cognitive function was finished. All protocols were conducted according to the guidelines for the care and use of laboratory animals. The Zhejiang Chinese Medicine University permitted this research.

The secretion of inflammatory cytokines in SAE mice’s brain tissues after administrating with UC-MSCs was determined by ELISA assay. After euthanasia of mice, brain tissues of mice were collected to measure the production of interleukin 6 (IL-6), tumor necrosis factor-$\alpha$ (TNF-$\alpha$), interleukin 1 beta (IL-1$\beta$) and high mobility group box 1 (HMGB1) using corresponding mouse ELISA kits following the protocols of the manufacturers. All ELISA kits were provided by R&D Systems (R&D Systems, Inc., Minneapolis, MN, USA) except for the HMGB1 ELISA kit (Biocompare, California, USA). For detection of IL-6, IL-1$\beta$ and TNF-$\alpha$, 50 $\mu$L of Assay Diluent RD1N and 50 $\mu$L of each sample or standard were mixed into the wells of a microplate, and incubated for 2 h at room temperature. Then, the wells were rinsed with Wash Buffer for 5 times, and 100 $\mu$L of Mouse IL-6, IL-1$\beta$ or TNF-$\alpha$ Conjugate were added to each well, incubated for 2 h. The wells were washed again for 5 times and each well was incubated in 100 $\mu$L of Substrate Solution for 30 min at room temperature in the dark condition. Finally, of the reaction was terminated by Stop Solution. The optical density of each well measured using microplate reader at 450 nm. For detection of HMGB1, 50 $\mu$L of each sample or standard were added into the wells of a microplate and incubated for 1 h at 37 ${}^{\circ }$C. After aspiration, 100 $\mu$L of Detection Reagent A was added to the wells and incubated for 1 h at 37 ${}^{\circ }$C. The wells were then washed for 3 times and 100 $\mu$L Detection Reagent B was added to the wells with incubation for 1 h at 37 ${}^{\circ }$C. After that, the wells were washed again for 5 times. Subsequently, 90 $\mu$L Substrate Solution was added to the wells and incubated for 20 min at 37 ${}^{\circ }$C, and 50 $\mu$L Stop Solution was added to the wells. The optical density of each well was measured using microplate reader at 450 nm.

Brain tissue cell lysate was isolated with RIPA lysis reagent (Beyotime Biotechnology, Shanghai, China). The lysate concentration was quantified by the BCA kit (Beyotime Biotechnology, Shanghai, China). The lysate was then subjected to SDS-PAGE and transferred to the PVDF membrane. Subsequently, the PVDF membranes were blocked with 5% non-fat milk and probed with primary antibodies including anti-p-NF-$\kappa$B (1:500), NF-$\kappa$B (1:1000), Iba1 (1:1000), Cleaved caspase-3 (1:500), Bax (1:1000), BCL-2 (1:1000), p-PI3K (1:2000), PI3K (1:1000), p-AKT (1:1000), AKT (1:500) and $\beta$-actin (1:5000) antibodies (Abcam, Cambridge, MA, UK) overnight at 4 ${}^{\circ }$C. The membranes were then incubated with secondary antibody IgG H&L (HRP) (Abcam, Cambridge, MA, UK) for 1 h at room temperature. Protein bands were shown utilizing Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, Piscataway, NJ, USA). The bands were visualized using ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA) and quantified using Image J software (National Institutes of Health, Bethesda, MD, USA). $\beta$-actin was designated as the control protein.

Cell apoptosis of cerebral cortex in SAE mice after administration with UC-MSCs was detected using TUNEL assay. The Click-iT${}^{\text{TM}}$ Plus TUNEL Assay (Thermo Fisher Scientific, Waltham, MA, USA) was applied to label fragmented DNA in the nucleus following the manufacturer’s protocol. DAPI was used to stain the nuclei of cells, which were then were observed and photographed using a fluorescence microscope (Carl Zeiss Inc., Jena, Germany).

Morris water maze test was carried out to evaluate mice’s neurological dysfunction as previously described [14, 15, 16]. The water maze consisted of a circular black pool (100 cm diameter, 38 cm deep), and it was filled with opaque water (25 cm deep). The water was prepared as black through adding non-toxic pigment, and the temperature of the water was kept at 23 $\pm$ 1 ${}^{\circ }$C. Then, a submerged (1.5 cm beneath the water surface) platform was placed into one pool quadrant. All mice underwent three training sessions for 5 days and a probe trial on the sixth day. All mice received four trials with a 15-min interval during each session. To perform the Morris water maze test, the mice were placed in the water facing the wall. All mice possessed 60 seconds to arrive at the platform. The mice were guided to the platform and stayed for 15 s. During the test, the latency to find the platform, the times of mice crossing the platform and swimming speeds were recorded.

HE staining was conducted to assess cerebral cortex neuron injury in SAE mice after administration of UC-MSCs. After euthanasia of mice, brain tissues of mice were collected to detect neuron damage of the cerebral cortex. Tissue samples were fixed in 10% formalin and embedded into paraffin. Subsequently, 4 $\mu$m-thickness sections were prepared. After deparaffinization and hydration, the sections were stained with hematoxylin and eosin for 5 and 3 minutes, respectively. The staining sections were observed under a light microscope (Carl Zeiss Inc., Jena, Germany).

To evaluate blood-brain barrier (BBB) integrity, mice in the different groups were collected to conduct Evans Blue staining. After mice were anesthetized, 2% Evans Blue (3 mL/kg) in sterile saline solution was injected into the tail vein of mice. After 1 h circulation, mice were transcardially perfused with cold saline. After that, mice were sacrificed, and the brain tissues were removed and weighed. The tissues were then prepared as homogenate, which was centrifuged for 20 min at 10000 g. After centrifugation, the supernatant was collected and the absorbance was determined at 620 nm.

All data were presented as mean $\pm$ standard deviation (SD), and data was analyzed by SPSS Statistics 22.0 (SPSS, Chicago, IL, USA). The differences among different groups were determined using one-way ANOVA. p 0.05 indicated a statistically significant difference.

The production of IL-6, IL-1$\beta$, TNF-$\alpha$, and HMGB1 were significantly increased in brain tissues of SAE mice, while which was decreased by UC-MSCs (p 0.01, Fig. 1A). Besides, UC-MSCs also inhibited the LPS-induced expression of phosphorylated NF-$\kappa$B (p 0.01, Fig. 1B). Furthermore, UC-MSCs suppressed the increase of microglia activation related biomarker Iba1 expression in brain tissues of SAE mice (p 0.01, Fig. 1C).

Fig. 1.

UC-MSCs inhibited the production of inflammatory factors in SAE mice. (A) The productions of IL-6, IL-1$\beta$, TNF-$\alpha$, and HMGB1 in brain tissues of UC-MSCs-treated SAE mice were determined using ELISA assay. (B) The protein levels of phosphorylated NF-$\kappa$B and total NF-$\kappa$B in brain tissues of UC-MSCs-treated SAE mice were detected using Western blot. (C) The protein level of microglia activation related biomarker such as Iba1 in brain tissues of UC-MSCs-treated SAE mice was determined using Western blot. **: p 0.01 means significant difference vs. sham group. ##: p 0.01 means significant difference vs. LPS group.

Cell apoptosis was remarkably induced by LPS in the cerebral cortex of SAE mice, which was inhibited by UC-MSCs treatment (p 0.01, Fig. 2A). Furthermore, apoptosis-related proteins including cleaved caspase-3 and Bax were increasedly expressed, while Bcl-2 was decreased in SAE mice, which was reversed by UC-MSCs treatment (p 0.01, Fig. 2B).

Fig. 2.

UC-MSCs inhibited cell apoptosis of the cerebral cortex in SAE mice. (A) Cell apoptosis of cerebral cortex in UC-MSCs-treated SAE mice was evaluated using TUNEL assay. (B) The protein levels of cleaved caspase-3, Bax, and Bcl-2 were detected by Western blot. **: p 0.01 means significant difference vs. sham group. #: p 0.05 means significant difference vs. LPS group. ##: p 0.01 means significant difference vs. LPS group.

The average body weight was significantly decreased in SAE mice compared to sham mice (p 0.01), which was restored by UC-MSCs treatment (p 0.05, Fig. 3A). Then, the cognitive dysfunction of SAE mice was explored using the Morris water maze test. Results showed that the latency to find the platform was increased in SAE mice compared to sham mice (p 0.01), while UC-MSCs administration decreased it (p 0.05, Fig. 3B). Besides, times of crossing the platform were decreased in SAE mice compared to sham mice (p 0.01), which was reversed by UC-MSCs treatment (p 0.05, Fig. 3C). Furthermore, the swimming speeds were decreased in SAE mice compared to sham mice (p 0.01), which was improved by UC-MSCs treatment (p 0.05, Fig. 3D). The movement route of mice in each group was presented in Fig. 3E.

Fig. 3.

UC-MSCs alleviated the cognitive dysfunction of SAE mice. (A) The average body weight of mice in sham, LPS, and LPS+ UC-MSCs groups was determined. (B) The latency to find the platform of mice in sham, LPS, and LPS+ UC-MSCs groups were recorded during the Morris water maze test. (C) Times of crossing the platform of mice in sham, LPS, and LPS+ UC-MSCs groups were recorded during Morris water maze test. (D) The swimming speed mice in sham, LPS, and LPS+ UC-MSCs groups were determined during Morris water maze test. (E) The movement routes of mice in sham, LPS, and LPS+ UC-MSCs groups were recorded during the Morris water maze test. **: p 0.01 means significant difference vs. sham group. #: p 0.05 means significant difference vs. LPS group.

The cerebral cortex of sham mice presented a normal histological structure with regular architecture and clear boundary (Fig. 4A). In SAE mice, cerebral cortex neuron injury was observed, characterized by condensed and hyperchromic nuclei, smaller cell bodies with perineuronal vacuolations around the degenerative neurons (Fig. 4A). However, UC-MSCs administration improved the abnormal morphology of the cortex and decreased the number of degenerated neurons (Fig. 4A). Moreover, Evans Blue leakage detection experiment was conducted to evaluate BBB integrity. It was observed that Evans Blue leakage was increased in SAE mice (p 0.01), which was decreased by UC-MSCs treatment (p 0.05, Fig. 4B).

Fig. 4.

UC-MSCs alleviated the cerebral cortex neuron injury of SAE mice. (A) HE staining was conducted to assess cerebral cortex neuron injury of UC-MSCs-treated SAE mice. (B) BBB integrity was assessed by Evans Blue leakage detection experiment. The black arrows indicated damaged neurons. **: p 0.01 means significant difference vs. sham group. ##: p 0.01 means significant difference vs. LPS group.

The phosphorylation of PI3K and AKT were decreased in SAE mice (p 0.01, Fig. 5). However, UC-MSCs administration rescued the decrease of phosphorylated PI3K and AKT (p 0.05, Fig. 5).

Fig. 5.

UC-MSCs activated PI3K/AKT pathway. The protein levels of p-PI3K, p-PI3K, p-AKT, and AKT in brain tissues were detected using Western blot. **: p 0.01 means significant difference vs. sham group. #: p 0.05 means significant difference vs. LPS group. ##: p 0.01 means significant difference vs. LPS group.