The characterization of hUC-MSCs
Healthy umbilical cord aspirates were used to isolate hUC-MSCs. The stem cells used in our experiment were donated by the Changchun Sigma Company and passaged by us. Under inverted microscopy, the morphology of hUC-MSCs was found to be spindle-shaped and fibroblast-like (Fig. 1a). If allowed to continue to grow, hUC-MSCs can present a whirlpool. To prevent senescent or differentiation of hUC-MSCs in the later passages, we used hUC-MSCs between passages 3 and 6 throughout our study. The characterization by flow cytometry confirmed the absence of CD45, CD34, CD14, CD11b, and HLA-DR− in hUC-MSCs, while they expressed CD29, CD105, CD73, CD166, and CD44 (Fig. 1b). This marker profile was consistent with those reported previously [7–10]. The cultures were observed for fuchsia bone nodules (Fig. 1c) and red lipid droplets formation (Fig. 1d). As evidenced by experimental results, hUC-MSCs could differentiate into the adipogenic and osteogenic lineages in vitro. In summary, the “hUC-MSCs” we obtained are consistent with most literature, indicating that the cells were hUC-MSCs with the ability to differentiate into multiple directions.
hUC-MSCs can improve the function of BEAS-2B and the enhancement effect of HP-hUC-MSCs
We established an in-direct contact co-culture system of BEAS-2B and hUC-MSCs or HP- hUC-MSCs to estimate the impact of hUC-MSCs on BEAS-2B. The Transwell chamber was suspended and used with 6-well plates. The upper layer was seeded with 1 mL (5 × 105 cells/well) of hUC-MSCs or HP-hUC-MSCs, and the lower layer was seeded with 2 mL (1 × 106 cells/well) of BEAS-2B, with the semipermeable membrane separated (Fig. 2a). BEAS-2B is a normal human bronchial epithelial cell that grows adherently and is epithelioid and polygonal (Fig. 2b).
In the LPS and co-culture groups, some BEAS-2B cells died in the periphery, while others were shrunken, with rough and granular material deposition, indicating that the BEAS-2B was damaged by LPS. We then used the CCK8 to detect the vitality of BEAS-2B. We found that exposure to LPS reduced the BEAS-2B vitality, which was reversed by hUC-MSCs (****, p < 0.0001), while HP-hUC-MSCs showed a stronger effect relative to the normal hUC-MSCs group (****, p < 0.0001) (Fig. 2c).
After LPS, sTREM-1 and TNF-α increased in the cell culture supernatant when compared with that in the control group, while IL-10 reduced. Different from the LPS treatment group, the extent of anti- inflammatory factor IL-10 in the co-culture group increased, while the inflammatory factors sTREM-1 and TNF-α decreased. Compared with the normal hUC-MSCs co-culture group, HP-hUC-MSCs increased IL-10 and decreased sTREM-1 and TNF-α to a relatively greater extent. These observations signify that co-culture with hUC-MSCs could reduce the BEAS-2B injury from LPS, while HP-hUC-MSCs had a greater impact on relieving the harm of BEAS-2B (Fig. 2d).
In comparison with the control group, the secretion of VEGF,NGF, KGF, and HGF in the LPS group was obviously different. When contrasted with the LPS group, the level of VEGF, NGF, KGF, and HGF in the normal hUC-MSCs co-culture group has obvious statistical difference. In accordance with the normal hUC-MSCs co-culture group, a visible difference was showed in the expression of VEGF, NGF, KGF, and HGF in the HP-hUC-MSCs co-culture group (Fig. 2e).
When compared with the control group, BEAS-2B showed a higher apoptosis in LPS group, which was manifested as an increased expression of apoptotic proteins PI3K, AKT, p-AKT, c-caspase3, and lowered level of Bcl-2, an anti-apoptotic protein. hUC-MSCs could alleviate apoptosis of BEAS-2B, which was manifested as a decreased expression of apoptotic proteins PI3K, AKT, p-AKT, and c-caspase3 and Bcl-2, an anti-apoptotic protein, expressed at a higher level. Furthermore, the reversal and repair of BEAS-2B damage in the HP-hUC-MSCs experimental group were found to be stronger than that in the normal hUC-MSCs test group (Fig. 2f).
Establishment of an LPS-mediated ALI mouse model
LPS is generally applied to build ALI model. In the ALI model mice, the lung volume increased, with obvious edema and focal hemorrhage on the lung surface. Under the microscope, infiltration of a large number of inflammatory cells was noted; the pulmonary interstitium widened; the alveoli collapsed, and the alveolar cavity exuded. Pulmonary capillary congestion, hemorrhage, and other pathological changes mainly manifested as inflammatory cell infiltration and pulmonary interstitial edema (Fig. 3a-b). Furthermore, stimulated neutrophils were discovered to have a critical role in initiating the proinflammatory processes that lead to hemorrhage or alveolar injury .
Other than in the lungs, no inflammatory cell infiltration was noted in the heart, liver, spleen, and kidney in the ALI model by LPS. The results showed that the inflammatory lesion is limited to the lungs (Fig. 3c). Therefore, our results prove that the intratracheal construction of this ALI model by LPS was feasible.
hUC-MSCs could improve ALI mice and the enhancement effect of HP-hUC-MSCs
We first weighed the dry and wet (not shown in the figure) weight of the lungs. Then, the W/D value was calculated to evaluate the extent of lung edema in each mice group. We also examined the MPO of the lung tissues to assess the degree of inflammatory cell infiltration and migration. Our results suggest that the W/D and MPO increased significantly after LPS stimulation. After the administration of normal hUC-MSCs, the W/D and MPO of the lung evidently decreased, showing a statistical difference. These factors decreased further, showing a statistically significant difference after the administration of HP-hUC-MSCs (Fig. 4a-b).
BALF is an important marker for evaluating lung functions, while the number of neutrophils in BALF could directly reflect the inflammatory changes in the mice. To further appraise the extent of inflammation in the mice pulmonary tissues, we determined the protein concentration in BALF by BCA in order to reflect the permeability of endothelial cells and epithelial cells. The neutrophil number and protein content of BALF increased significantly with LPS stimulation, with an increase in the permeability of the endothelial and epithelial cells. After the administration of normal hUC-MSCs, the neutrophil number and protein concentration BALF reduced, indicating alleviation of inflammation in the experimental mice. After the administration of HP-hUC-MSCs, the neutrophil number and protein concentration in BALF reduced significantly. The inflammation in the lung was further reduced, indicating a significant statistical difference (Fig. 4c-d).
In the control group, the lungs of the mice were well inflated, and there was no hemorrhage on the surface. At each time point for the ALI model mice, the lung volume increased with an obvious edema, and focal hemorrhages were detected on the lung surface. The microscopic observation revealed the infiltration of a large number of inflammatory cells, widening of the pulmonary interstitium, the collapse of the alveoli, pulmonary capillary congestion, hemorrhage, and other pathological changes, which manifested in ALI treatment group. By contrast, the normal hUC-MSCs treatment group showed less edema, bleeding, and congestion. The HP-hUC-MSCs experiment group highly reduced above pathological changes. No statistical difference between the CoCl2 preconditioned hUC-MSCs group and hypoxia incubator-treated hUC-MSCs group (Fig. 4e).
After LPS, the levels of sTREM-1 and TNF-α in BALF increased relative to those of the control group, while the level of IL-10 decreased. After transplantation of hUC-MSCs, the level of IL-10 in BALF increased, while the concentration of sTREM-1 and TNF-α decreased, which together indicated that the inflammation in the ALI model mice was alleviated by hUC-MSCs. hUC-MSCs exhibited a repairing effect on the ALI mice. The HP-hUC-MSCs treatment group significantly improved the above-mentioned inflammatory and anti-inflammatory factors. Furthermore, serum concentrations of sTREM-1, TNF-α, and IL-10 followed the same trend as BALF, although the absolute concentration was significantly lower than those in BALF, implying that the inflammatory injury in other parts was much lighter than that in the lungs. This finding further validates the feasibility of our method to construct the ALI model using LPS (Fig. 4f).
In the LPS model group, the apoptosis of lung tissue increased, as manifested by increased expression of apoptotic proteins AKT, p-AKT, and PI3K, along with a reduction in the anti-apoptotic protein Bcl-2 expression. The normal hUC-MSCs could reverse the apoptosis of mouse lung tissues, as manifested by a decrease in the expression of the apoptosis proteins AKT, p-AKT, and PI3K, but an increase in the Bcl-2 expression, along with the reversal and repair effect of HP-hUC-MSCs on the mouse lung tissues that was stronger than that of normal hUC-MSCs. In addition, the changes in these genes in qPCR revealed the same trend in Western blotting. According to the findings, this process could be linked to the PI3K/AKT pathway. (Fig. 4g-h).
In LPS-induced ALI mice, hUC-MSCs can suppress TREM-1 expression
Our results confirmed that hUC-MSCs could limit TREM-1 expression in ALI mice mediated by LPS from 3 aspects of Western blotting, IHC, and qPCR, which has a significant impact on the the treatment of ALI.
In the IHC, 5 high-power fields (×400) were randomly selected from each section. Then, the area of positive objects under every high-power field was calculated utilizing Image-J software, reflecting the intensity of positive protein. As can be seen from the figure, the inflammatory cells including neutrophils, monocytes, and macrophages in the LPS group were strongly TREM-1 positive; the hUC-MSCs group was TREM-1 positive for a few scattered inflammatory cells, and the HP-hUC-MSCs group was positive for individual inflammatory cells. Moreover, no obvious difference was seen between the CoCl2-pretreated hUC-MSCs group and hypoxia incubator-treated hUC-MSCs group (ns, p > 0.05) (Fig. 5a).
The level of TREM-1 in lung was dramatically elevated after LPS stimulation, according to Western blotting and qPCR data. (####, p < 0.0001). After the administration of normal hUC-MSCs, TREM-1 expression was significantly reduced, showing a statistical difference (****, p < 0.0001); after the administration of HP-hUC-MSCs, the expression of TREM-1 was further decreased, demonstrating that the difference is statistically significant (****, p < 0.0001) (Fig. 5b-d).
Individuals with severe pneumonia had significantly higher concentration of sTREM-1 in their serum
A sample of 40 individuals were hospitalized to Jilin University's Second Hospital of the Jilin University from June–December 2021 enrolled in this study. The 24 patients with severe pneumonia included 13 men and 11 women of an average age of 67 ±14 years. The 16 patients of common pneumonia included 10 men and 6 women of an average age of 64 ±5 years. The 10 healthy controls included 4 men and 6 women of an average age 62 ±4 years. No significant difference was noted in the age and gender among the 3 groups (p > 0.05). This study was conducted after obtaining informed consent from the patients before blood collection. (Fig. 6)
hUC-MSCs protect ALI mice from lung injury by inhibiting TREM-1 expression.
To achieve the dry weight, we first determined the lungs' wet weight and then baked them for 48 hours at 60°C. The W/D value was calculated to determine the severity of pulmonary edema in each group of mice. We also examined the MPO of the lung tissues to assess the degree of inflammatory cells infiltration and migration. The results indicated that the W/D and MPO of the lungs increased significantly in the LRS + LPS group. LR12 is a synthetic inhibitor of TREM-1. After the administration of LR12 or normal hUC-MSCs, the W/D and MPO of the lung evidently decreased, showing a statistical difference. This level decreased further, and a statistically significant difference was noted after the administration of LR12 and normal hUC-MSCs. The results showed that hUC-MSCs could reduce the W/D and MPO of ALI mice by inhibiting the expression of TREM-1 (Fig. 7a-b).
BALF is an important marker for evaluating lung functions, while the total number of cells in BALF, particularly neutrophils, directly reflected the inflammatory changes in mice. To get a better idea of how much inflammation there is in mice's lungs, we determined the protein concentration in BALF by BCA to reflect the permeability of the endothelial and epithelial cells. In BALF, the number of neutrophils, all cells, and protein content all rose significantly in the LRS + LPS group along with an increase in the permeability of endothelial and epithelial cells. After the administration of LR12 or normal hUC-MSCs, the neutrophil number, overall cells, and protein content in BALF lowered, indicating improvement in inflammation in the experimental mice. After the administration of LR12 and normal hUC-MSCs, the neutrophil number, complete cells, and protein concentration in BALF diminished significantly. The inflammation in the lungs reduced further, and there was a significant statistical difference. Our results showed that hUC-MSCs could reduce the neutrophil number, cumulative cells, and protein content in BALF by inhibiting the expression of TREM-1 (Fig. 7c-e).
The mice's lungs were well inflated in the control mice, and there was no hemorrhage on the surface. At each time point in the LRS + LPS group, the lung volume increased with obvious edema, and focal hemorrhages were noted on the lung surface. The microscopic observation showed the infiltration of a large number of inflammatory cells, widened pulmonary interstitium, collapsed alveoli, pulmonary capillary congestion, hemorrhage, and other pathological changes mainly manifested in the LRS + LPS mice. In contrast, the normal hUC-MSCs or the LR12-treatment group showed less edema, bleeding, and congestion. The normal hUC-MSCs and LR12 treatment group significantly reduced the abovementioned pathological changes. As noted above, results indicated that by decreasing TREM-1 expression, hUC-MSCs alleviated ALI mice with lung inflammation. (Fig. 7f).
In IHC, 5 high-power fields (×400) were randomly selected from each section. The area of positive objects under every high-power field was calculated using Image-J application, reflecting the intensity of positive protein. As can be seen from the figures, the inflammatory cells, including neutrophils, monocytes, and macrophages, in the LRS + LPS group were strongly TREM-1 positive; the LR12 + LPS group and LRS + LPS + hUC-MSCs group was TREM-1 positive for a few scattered inflammatory cells, and the LR12 + LPS + hUC-MSCs group was positive for individual inflammatory cells. These values indicated that the administration of LR12 or hUC-MSCs could significantly diminish TREM-1 expression in ALI mice. Moreover, LR12 and hUC-MSCs could synergistically suppress TREM-1 expression of in ALI mice (Fig. 7g).
In the LRS + LPS group, the levels of sTREM-1 and TNF-α in the lung tissues, serum, and BALF risen relative to those in the control mice, albeit IL-10 level declined. After the transplantation of hUC-MSCs or LR12, IL-10 concentration in lung tissues, serum, and BALF increased, while the concentration of sTREM-1 and TNF-α decreased, indicating that the inflammation was alleviated by hUC-MSCs or LR12. hUC-MSCs or LR12 exhibited a repairing effect on ALI mice. The LR12 + LPS + hUC-MSCs group significantly improved the abovementioned inflammatory and anti-inflammatory factors. These results also indicate that by reducing TREM-1 expression, hUC-MSCs were able to reduce lung inflammation in ALI animals. Further, Zhu J Q et al. found that MSCs reduce ALI damage generated by LPS-induced by suppressing Ly6C + CD8 + T cells' pro-inflammatory activity . (Fig. 7h).
The therapeutic impact of hUC-MSCs in mice with LPS-caused ALI was associated with TREM-1-regulated TLR4 and MyD88
When comparison to the control group, TREM-1, TLR4, and MyD88 expression in the LRS + LPS mice were greatly upregulated (p < 0.001). When compared to the LRS + LPS mice, the LR12 + LPS and LRS + LPS + hUC-MSCs groups showed inhibition in TREM-1, TLR4, and MyD88 expression, showing an obvious statistical difference (p < 0.001). By contrast, TREM-1, TLR4, and MyD88 expression levels were considerably lower in the LR12 + LPS + hUC-MSCs group (p < 0.05), demonstrating that TREM-1-mediated expression of TLR4 and MyD88 was relevant to the protective role of hUC-MSCs against ALI (Fig. 8a-b).
The therapeutic impact of hUC-MSCs in mice with LPS-caused ALI was connected to TREM-1-regulated PI3K/Akt phosphorylation
When compared with the control group, the expressions of p-PI3K and p-Akt in the LRS + LPS group upregulated, and there was a statistical difference (p < 0.05). When contrasted with the LRS + LPS mice, LR12 + LPS and LRS + LPS + hUC-MSCs groups showed significant inhibition in p-PI3K and p-AKT expression, and displayed an obvious statistical difference between them (p < 0.001). In contrast, the LR12 + LPS + hUC-MSCs group exhibited a stronger effect on reducing p-PI3K and p-AKT levels, demonstrating a substantial distinction among them (p < 0.05). These results indicated that the preventive impact of hUC-MSCs on LPS-caused ALI correlated with PI3K/Akt phosphorylation level through TREM-1 (Fig. 9).