Differences in appearance and lung tissue structure between the young and elderly macaques
Elderly macaques were found to have a dull coat that turned white, especially around the head and face. Besides, their skin was loose and dry, and the face red (Fig. 1).
The lung tissue of the young control group and the elderly model group were soft, butterfly-shaped, flexible, and pale red. However, when compared with the young control group, the lung size of the elderly model macaque was significantly larger than that of the young control (Fig. 2).
The young control group showed clear lung structures, thin and smooth alveolar walls, no thickening of the alveolar space, no exudate, and only a small amount of inflammatory cellular infiltrate around the blood vessels. In the elderly model group, although the alveolar wall thickness was uniform and the alveoli clean, there was no exudate, the alveolar cavity became irregularly enlarged to form the pulmonary bullae and there was visible pigmentation (as indicated by the black arrow). The elderly model group showed severe inflammation compared with the young control group. The average area and MLI were significantly increased in the elderly model group compared to the young control group (Fig 3), (P <0.0001).
Masson’s Trichrome stain colored the collagen fibers blue. The collagen area in the lungs of the elderly model group was significantly increased, compared with the young control group (Fig .4),(P <0.01).
Immunohistochemical staining to label the vascular endothelial cells with CD31 revealed that the lung tissue cells nuclear were stained blue, and the surface markers of vascular endothelial cells CD31 were stained brown. In the elderly group, the expression of CD31 in the lungs was significantly reduced compared with the young control group (Fig .5), (P<0.0001).
Cultivation and identification of BMMSCS
Macaque BMMSCSs were isolated from bone marrow aspirates and cultured by adherent culture screening. A few fusiform adherent cells were observed under an inverted phase-contrast microscope after 3-4 days. The cell fusion rate had reached about 80% after 9 days. BMMSCSs of passage 3 to 5 showed uniform morphology, they grew densely spiral and were isolated (Fig .6A).
To confirm the purity of the cultured cells, the immunophenotypes of the P4 generation of juvenile macaque BMMSCS were analyzed by flow cytometry. A panel of surface antigens was analyzed. The results showed that the BMMSCSs were positive for CD29, CD45, CD73, CD90 and CD184 at percentage rates of 96.35 ± 0.62, 0.16 ± 0.12, 95.22 ± 0.37, 96.25 ± 1.71, 93.53 ± 2.76, respectively (Fig .6B,Table 1).
Table 1 Flow cytometry analysis of surface antigens
Surface antigen
|
n
|
Cell positive rate
|
CD29
|
3
|
96.35±0.62
|
CD45
|
3
|
00.16±0.12
|
CD73
|
3
|
95.22±0.37
|
CD90
|
3
|
96.25±1.71
|
CD184
|
3
|
93.53±2.76
|
n is the number of repeated experiments
The proliferation assay showed that the BMMSCSs took on an “S” shape, the cells remained latent for the first 1-2 days, and entered a logarithmic proliferation phase on 3 to 7 days, where the cells grew vigorously and had the best vitality. On the 8th day, they entered the plateau phase which was characterized by a reduction in proliferation (Fig .6C).
The P4 generation of young macaque BMMSCS was used to determine the differentiation ability and proliferation in vitro. The duration of the differentiation experiment was 14-21 days. The cells were cultured in osteogenic induction medium and allowed to aggregate, form nodules, and accumulate calcium deposits. Alizarin red stain was used to detect the precipitated calcium deposits which were an indication of differentiation. Intracellular lipid droplets were stained with oil red O, and red-stained lipid droplets were found in the cells. Proteoglycans were stained with Alcian blue and appeared as smears (Fig .6D).
The Changes in lung tissue structure after BMMSCS treatment
PET-CT examination was performed on the macaques, before treatment, at 90 and 180 days after treatment. The results showed that the texture of the lungs of the macaque before treatment was grid-like, ground-glass opacity, honeycomb-shaped, with the peripheral, subpleural, and lower lung lobes as the main features and emphysema was obvious. The average Hounsfield unit was significantly decreased before treatment compared with the control group (P <0.01). Before treatment, HRCT showed irregular thickening of the leaflet intervals, and the small blood vessels in the leaflet became obvious due to the thickening of the wall in the treatment group. PET showed that the 18F-FDG uptake quantified as the glucose uptake in the lungs decreased after treatment. At 90 and 180 days, following treatment, CT showed that the lungs' texture was clear, and both hilar were normal. The Hounsfield unit was higher than before treatment. The average Hounsfield units were-(685 ± 12.53) and-(705 ± 18.53), respectively (Table 2,Fig .7).
Table 2 Changes in PET-CT in elderly macaque lung after BMMSCS treatment
|
n
|
Hounsfield Unit
|
SUV max
|
Control
|
5
|
-(672±12.52)
|
0.4±0.09
|
Prior treatment
|
5
|
-(853±25.32)$
|
0.7±0.06 $
|
90 days after treatment
|
5
|
-(685±12.53)*
|
0.5±0.08 *
|
180 days after treatment
|
5
|
-(705±18.53)*
|
0.3±0.07 **
|
n is for the number of animals analyzed, $P <0.05 when compared with the control group,* P <0.05 when compared with prior treatment, ** P <0.01 when compared with the prior treatment
Examination of material from the lung tissue after 180 days of treatment, showed that the lung tissue appeared dark white and red without embolism. However, there were no significant changes observed in both the treatment group compared with the model group (Fig .8).
HE results showed that the control group had clear lung structures, thin and smooth alveolar walls, no thickening of the alveolar spaces, no exudates, and only a small amount of inflammatory cellular infiltration around the blood vessels. The alveolar cavity showed irregular enlargement and the formation of pulmonary bullae,the alveoli were clean with no exudates seen, a small amount of inflammatory cell infiltration was observed around the blood vessels with pigmentation in the model group macaques and the treatment group macaques. Although the inflammation score was not statistically different in those groups, the treatment group had lower inflammation than the model group. The average alveolar area and alveolar lining interval (MLI) of the model group and the treatment group were both significantly increased compared with the control group,(P <0.05); however, there was no significant difference between the model group and the treatment group (Fig .9),( P> 0.05).
Masson’s Trichrome stain showed blue collagen. The collagen area of the treatment group was significantly reduced (P <0.05) when compared with the model group (Fig .10).
To determine the changes in capillary density around the alveoli after cell transplantation, immunohistochemistry was performed using CD31 as a marker of vascular endothelial cells. The nucleus stained blue and capillaries with CD31 surface markers were stained brown. The content of CD31 around the alveoli was significantly increased in the treatment group, compared with the model group (Fig .11), (P<0.0001).
Effect of BMMSCS on senile type 2 alveolar epithelial cells
Type Ⅱ alveolar epithelium plays a significant role in lung aging. In the elderly, the quantity and quality of type Ⅱ alveolar epithelial cells are significantly reduced [15]. In this study, the effect of BMMSCS on lung structure was observed using type Ⅱ alveolar epithelial cells to explore the specific effect on lung cells. Hydrogen peroxide was used to establish an aging model of A549 cells. Different concentrations of hydrogen peroxide were found to induce different degrees of aging in A549 cells. Following SA-β-gal staining, we found that when the hydrogen peroxide concentration was 600μm/L and 800μm/L, the senescence rate of A549 cells was the highest. In addition, 1000μmol / L and 1200μmol / L were not considered as most of the cells were apoptotic and deformed(Fig 12).
RT-PCR was used to detect the expression of P53 gene in A549 cells after induction of aging. The increase in P53 expression was most significant (P <0.001, P <0.0001) at 600μmol / L, 800μmol / L hydrogen peroxide concentration (Fig 13A). Therefore, 600μmol/L was chosen as the optimal concentration to induce senescence of A549 cells.
RT-PCR was used to further compare the expression of P53, P21, TERT, TCAB1 before and after induction of aging using hydrogen peroxide at a concentration of 600 μmol / L. At 6h, after induction, the changes in P53 and P21 expression were significantly increased (P <0.0001, P <0.001); however, TERT and TCAB1 were significantly decreased (P <0.001, P <0.01) 6 hours after induction (Fig .13B). At 24h,48h, and 72h after changing the medium, TCAB1 did not show any significant change (P> 0.05), while P21 was significantly increased (P <0.0001). Even though the expression of P53 was significantly decreased after induction (P<0.05), it remained higher than before induction (Fig .13C).
Following the indirect co-culture of A549 cells aging model with BMMSCS for 48H, the lower layer of A549 cell was collected to explore the effect of BMMSCS on the A549 aging model by RT-PCR. The expression levels of P53 and P21 were found to be significantly decreased (P <0.001, P <0.01) in the treatment group compared with the model group. However, the expression level of TCAB1 increased significantly (P <0.05) (Fig .14A).
The ROS level, apoptosis ratio, and cell cycle of A549 cells were detected by flow cytometry after indirect co-culture. The ROS level and apoptosis ratio of the treatment group were found to be significantly reduced compared with the model group (Fig .14B, Fig .14C), (P < 0.0001, P <0.001). Proliferation in the treatment group accelerated to the G2 phase (Fig .14D), (P <0.01).
In vitro experiments, revealed the effects of BMMSCS on the aging of the A549 cell model. To verify these effects in vivo, proSPC was used as a marker for type Ⅱ alveolar epithelial cells. The results showed that type Ⅱ alveolar epithelial cells were round or oval and scattered in the alveolar wall. The number of type Ⅱ alveolar epithelial cells in the model group was significantly reduced compared with the control group (P <0.001). However, in the treatment group, typeⅡ alveolar epithelial cells were significantly increased compared with the model group (Fig .15),(P <0.01).
Changes in VEGF expression level in lung tissue
There were observed changes in the density of capillaries around the alveoli, hence there was a need to check on the level of VEGF in the lungs. Western blot analysis of the lung tissue showed that the VEGF in the model group decreased significantly compared with the control group (P <0.05). Besides, after BMMSCS treatment, the VEGF level in the treatment group was significantly higher compared with the model group (Fig .16) (P <0.05).
Changes in the level of ROS and inflammatory factors after BMMSCS treatment
Extensive experiments in a wide range of organisms from yeast to primates have revealed that the nine hallmarks of aging are stem cell failure, changes in intercellular communication, genomic instability and telomere wear, epigenetic changes, loss of protein homeostasis, nutrition changes, mitochondrial dysfunction and cellular senescence [16]. There are still many unresolved issues on the main causes and impacts of these events. However, emerging research suggests that the causes and commonalities of these events are related to the immune system. Inflammatory aging is characterized by elevated levels of immune cell infiltration and elevated levels of pro-inflammatory cytokines and chemokines in the tissue microenvironment and circulatory system [16]. Under normal physiological conditions, ROS in the cells is constantly generated and eliminated. Therefore, maintaining appropriate levels of ROS in the cells plays an important role in the stability of cell functions. However, in the state of aging, the level of ROS may also be elevated due to mitochondrial stress and damage and persistent inflammation [17]. High levels of ROS not only increase damage to the cells but also stimulates immune cells to produce more pro-inflammatory factors to form a vicious circle [18]. The immune regulation and damage repair functions of mesenchymal stem cells are very critical. Studies have reported that MSCs control inflammation and ROS production through paracrine and mitochondrial transfer between MSCs and aging cells [19, 20]. Therefore, this study proposed that mesenchymal stem cells altered inflammation and ROS levels in elderly macaques thus affecting lung degeneration. The frozen section of lung tissue was used to detect the level of ROS. Using the inverted fluorescent microscope, the lung cells' nucleus was stained blue, and the red fluorescence was distributed in the cytoplasm. Compared with the model group, the ROS level of the treatment group was significantly reduced (Fig .17),(P <0.01).
To explain the regulatory effect of BMMSCS on aging-related inflammation, the levels of IL-1β, IL-17A, and TNF-α were detected by ELISA. IL-1β was found to be significantly decreased in blood serum (P <0.05) compared with the model group at 30 and 60 days after BMMSCS treatment and returned to the original levels after 90 days. Besides, TNF-α was found to be significantly decreased (TNF-α) after 30 days (P <0.05), and returned to original levels after 60 days, and remained unchanged. There was no significant change in IL-17A levels (Fig .18A).
The changes in the levels of inflammatory factors including IL-1β, IL-6, TNF-α, and IL-10 in the lung after BMMSCS treatment were determined by western blot analysis. The levels of IL-1β, IL-6, TNF-α in the treatment group were significantly lower than in the model group. The level of IL-10 in model group was significantly lower than in the control group (P <0.05), but was significantly increased after BMMSCS treatment (Fig .18B), (P <0.05).
Effect of BMMSCS treatment on immune regulatory cells
As described above, there were observed changes in the expression levels of inflammatory factors in peripheral blood and lung tissue after BMMSCS treatment. Treg cells are a class of cells with immune-regulatory functions and also play a vital role in the regulation of inflammation. The ratio of Treg cells in the peripheral blood was measured by flow cytometry. The results showed that the Treg ratio in macaque peripheral blood decreased significantly at 30 days after BMMSCS treatment (P <0.01), and reached its minimum at 60 days after treatment (P <0.0001) compared with the model group. However, there was no significant change in the ratio of Treg cells at 60 and 90 days (Fig .19), (P> 0.05).
To determine whether the changes in Treg cells in the periphery and lung tissue were consistent, the Treg cell surface marker FOXP4 was used and detection was performed by immunohistochemistry. The content of FOXP4 in the lung tissue of the model group was found to be significantly higher compared with the control group (P < 0.01). Besides, the content of FOXP4 in the treatment group was significantly lower than that in the model group (P <0.0001), and the control group (Fig .20).