Human Amnion-Derived MSCs Alleviate Acute Lung Injury and Hinder Pulmonary Fibrosis Caused by Paraquat in Rats

Abstract

Methods: First, the purity of hAD-MSCs was determined by morphological observation and FCM, and the effects on the survival of paraquat-poisoned Sprague-Dawley rats were observed. All rats were randomly divided into three groups, defined as the sham control group (n = 8), model group (n = 15), and hAD-MSC-transplanted group (n = 17). Pneumonocyte damage and inflammatory cell infiltration were investigated in the three groups of rats, untreated control, paraquat only, and paraquat+hAD-MSC transplanted, using H&E staining. Fibrosis was investigated in three groups of rats using Masson's trichrome staining and Sirius red staining. The profibrotic factor TGF-β1, the composition of fibrotic collagen HYP, and the hAD-MSC-secreted immunosuppressive factor HLA-G5 in serum were investigated in the three groups of rats using ELISA. Furthermore, the distribution of hAD-MSCs was investigated in the three groups of rats using immunohistochemistry and hematoxylin staining.

Results: The hAD-MSCs exhibited typical hallmarks of MSCs, improved the state of being and survival of paraquat-poisoned rats, reduced both lung injury and inflammation, and inhibited the progression of pulmonary fibrosis by decreasing the deposition of collagen and the secretion of both TGF-β1 and HYP. The hAD-MSCs could survive in damaged lungs and secreted appropriate amounts of HLA-G5 into the serum.

Conclusion: The obtained results indicate that hAD-MSCs used to treat paraquat-induced lung injury may work through anti-inflammatory and immunosuppressive pathways and the downregulation of profibrotic elements. This study suggests that the transplantation of hAD-MSCs is a promising therapeutic approach for the treatment of paraquat-intoxicated patients.

Figure 1

Morphology of cultured hAD-MSCs and phenotypic analysis of purified hAD-MSCs by FCM. (a) Primary hAD-MSCs with diverse morphology. (b) Third-passage purified hAD-MSCs with fibroblast-like morphology. The scale is 200 μm. (c) Purified hAD-MSCs exhibited classic phenotypic hallmarks of MSCs, stained strongly positive for CD44, CD73, CD90, and CD105 and stained negative for CD14, CD19, CD34, CD45, and HLA-DR.

Figure 2

Survival analysis. All rats in the model group (n = 15) and transplanted group (n = 17) after PQ poisoning were observed daily for 7 days. The sham control group (n = 8) was observed at the same time, and the survival curves were drawn. Kaplan-Meier survival analysis using the log-rank (Mantel-Cox) test (^∗p < 0.05).

Figure 3

H&E staining of lung tissue. (a) Rats in the model group (n = 6) showed significant pulmonary interstitial thickening, mutual integration between alveoli, significant damage to the structure, disappearance of the original alveolar structure, a large number of interstitial lung telangiectasia, associated diffuse pulmonary hemorrhage, pulmonary capillary, and alveolar spaces surrounding visible inflammatory cell infiltration. Rats in the hAD-MSC-transplanted group (n = 13) also showed alveolar structural damage, fusion, a small amount of interstitial pulmonary capillary congestion, exudation around a small number of red blood cells, and pulmonary capillaries and alveoli around inflammatory cells. Inflammatory cell infiltration was significantly less than that of the model group. The scale is 100 μm under low magnification view and 50 μm under high magnification view. (b) Histologic scores were obtained blindly using previously published criteria [25]. Each bar represents the mean ± SEM (^∗∗p < 0.01, ^∗∗∗p < 0.001). Not marked if not significantly different.

Figure 4

hAD-MSCs hinder PQ-induced pulmonary collagen deposition. Masson's trichrome staining of lung tissue. Collagen fibers were dyed blue by Masson's trichrome stain. (a) The collagen deposition is significantly increased in the model group rats (n = 6) compared to the model group rats (n = 8) but reduced in the hAD-MSC-transplanted group rats (n = 13). The scale is 100 μm under low magnification view and 50 μm under high magnification view. (b) Collagen fibers were dyed blue and quantified by using ImageJ. ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001.

Figure 5

hAD-MSCs reduce PQ-induced pulmonary type I collagen deposition. Sirius red staining of lung tissue. (a) Red staining on light microscopy is type I collagen deposition, which is significantly increased in the model group rats (n = 6) compared to the model group rats (n = 8) but reduced in the hAD-MSC-transplanted group rats (n = 13). The scale is 100 μm under low magnification view and 50 μm under high magnification view. (b) Type I collagen fibers were dyed red and quantified by using ImageJ. ^∗p < 0.05, ^∗∗p < 0.01.

Figure 6

hAD-MSCs reduce the cytokine that promotes collagen formation and collagen fiber. TGF-β1 (a) and HYP (b) in serum were measured by ELISA. TGF-β1, a major profibrotic cytokine, and HYP, the main component of collagen fibers, were measured using an ELISA kit. TGF-β1 levels increased following PQ exposure, accompanied by the increased HYP serum concentration in the model group (n = 6) compared to the sham control group (n = 8). TGF-β1 and HYP levels decreased following hAD-MSC transplantation (n = 13). Statistical analysis was performed using the unpaired t-test. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. ns = not significant.

Figure 7

Survival of hAD-MSCs in the damaged lung and concentration of HLA-G5 in serum. (a) Human leukocyte antigen G5 was stained with antibody HLA-G5. Hematoxylin was used to redye cell nuclei. HLA-G5-positive cells were not observed in the lung from the control group (n = 8) and model group (n = 6), but the hAD-MSC-transplanted group (n = 13). The scale is 100 μm under low magnification view and 50 μm under high magnification view. (b) The HLA-G5-positive cells were quantified per 100 cells. (c) Secretion of the immune suppressor HLA-G5 in serum was measured using an ELISA kit. HLA-G5 in rats from the hAD-MSC-transplanted group (n = 13) was remarkably higher than that in the control group (n = 8) and model group (n = 6). There was almost no difference in HLA-G5 levels between the other two groups in serum. Statistical analysis was performed using the unpaired t-test. ^∗∗∗∗p < 0.0001. ns = not significant.