脑损伤
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Figure 3｜Neurological evaluations of rats at 1 month after hpcMSCs transplantation.
Before transplanting hpcMSCs, we conducted immunofluorescence staining to identify the cells. The cells expressed CD90+CD44+CD45–, consistent with MSC characteristics (Figure 3A). Subsequently, the hpcMSCs were transplanted three times into the HI rats at 1, 3, and 10 days after HI. At 1 month after hpcMSCs transplantation, behavioral tests including open field test, Morris water maze, Y-maze test, rotarod test, and NSS were used to evaluate the neurological functions of rats. The open field test showed that the number of times grooming, the time spent grooming, and the number of fecal particles were significantly increased in the HI group compared with those in the sham group, and these measures were significantly decreased in the hpcMSCs group compared with the NS group (P < 0.05; Figure 3B). Additionally, the time spent rearing was reduced in HI rats compared with sham rats, and rearing time in hpcMSCs rats was greater than that in NS rats (P < 0.05; Figure 3B). The number of rearing times showed consistent results.
During the first 5 days of training in the Morris water maze, the latency to target in the HI group was significantly longer than that in the sham group, and a significant decrease was observed in the hpcMSCs treatment group relative to the NS group (P < 0.05; Figure 3C). After removing the target at 6 days, the distance traveled in the target quadrant and the number of target crossings of HI rats were both reduced compared with those in the sham group (P < 0.05). These measures were significantly increased in the hpcMSCs treatment group compared with the NS group (P < 0.05; Figure 3C).
Y-maze test results showed that the number of food arm entries, the time spent in the food arms, and the accuracy rate were significantly decreased in the HI group compared with the sham group, and these measures were increased in the hpcMSCs treatment group compared with those in the NS group (P < 0.05; Figure 3D). Similarly, the measures in the error arm were significantly decreased in the hpcMSCs treatment group (P < 0.05; Figure 3D). Additionally, the decreased rotarod test time in the HI group was reversed after hpcMSCs treatment (P < 0.05; Figure 3E). The NSS showed that the neurological dysfunctions caused by HI were alleviated with hpcMSCs treatment (P < 0.05; Figure 3F). The behavioral evaluations indicated that hpcMSCs transplantation significantly improved neurological function including cognitive, locomotor, and anxiety-like behaviors in HI rats at 1 month.
Figure 6｜Double immunofluorescence staining of IL-3 and NeuN (A) or GFAP (B) in the right and left cortex and hippocampus.
Figure 7｜Downregulation of IL-3 impairs the viability and growth of SH-SY5Y cells after OGD.
Immunofluorescence staining showed that IL-3 was expressed in neurons (marked by NeuN) and astrocytes (marked by GFAP) of the ipsilateral cortex and hippocampus (Figure 6A and B). CY3-labeled SH-SY5Y cells indicated successful transfection with IL-3 siRNA (Figure 7A). RT-PCR showed that F2 significantly downregulated IL-3 expression and was the most efficient siRNA (P < 0.05; Figure 7B). Thus, F2 was selected for subsequent experiments. CCK-8 assay and MTT assay showed that cell viability and proliferation, respectively, were significantly reduced after IL-3 downregulation compared with those in the OGD + NC and OGD + reagent groups (P < 0.05; Figure 7C and D). Over time, the number of cells in the IL-3-si group was obviously less than that in the NC and reagent groups on days 1–6 (P < 0.05; Figure 7E).