The effects of BMMSC treatment on lung degeneration in elderly macaques

Age-related degeneration of lung tissues increases the risk of lung injury and exacerbates lung disease. It is also the main risk factor for chronic lung diseases (such as COPD, idiopathic pulmonary fibrosis, cancer, etc.). Here, we performed systematic screening, evaluation of elderly macaque model. A senile multiple organ dysfunction model was used to explored whether BMMSC could improve degeneration of lung tissues in an elderly macaque model. Using model evaluation tests, we found that the average alveolar area, Mean linear intercept （ MLI ） and fibrosis area in the elderly macaque models were significantly larger than in young rhesus monkeys (P <0.05), and the capillary density around the alveoli was significantly lower than in young macaque models (P <0.05). Intraveous infusion of BMMSC reduced the degree of pulmonary fibrosis in elderly macaque, increased the density of capillaries around the alveoli (P <0.05), and the number of type Ⅱ alveolar epithelium in elderly macaque (P <0.05). BMMSC infusion reduced lung tissue ROS level, systemic and lung tissue inflammation level and Treg cell ratio in elderly macaque model （ P ＜ 0.05 ） . Indirect co-cultivation revealed that BMMSC reduced the expression of senescence-related genes, ROS levels, apoptosis rate of aging type Ⅱ alveolar epithelial cells (A549 cells) and promoted their proliferation (P ＜ 0.05).


Fig .1 age-related change in appearance between the young control group and the elderly model group
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 volume 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).  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 BMMSC
Macaque BMMSCs 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. BMMSCs 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 P3 generation of juvenile macaque BMMSC were analyzed by flow cytometry. A panel of surface The proliferation assay showed that the BMMSCs 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 P3 generation of young macaque BMMSC 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    Masson staining showed blue collagen. The collagen area of the treatment group was significantly reduced (P <0.05) when compared with the model group (Fig .10).

Effect of BMMSC 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 [14] . In  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).

Changes in the level of ROS and inflammatory factors after BMMSC 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 [15] . 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 [15] . 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 [16] . 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 [17] . 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 [18,19] . 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 BMMSC 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 BMMSC 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 BMMSC 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 BMMSC treatment ( Fig .18B), (P <0.05). 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 FOXP3 was used and detection was performed by immunohistochemistry. The content of FOXP3 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 FOXP3 in the treatment group was significantly lower than that in the model group (P <0.0001), and the control group (Fig .20). High inflammation and oxidative stress are important factors driving degeneration of tissues and organs during aging [6,15,23] . In this study, we found high level of agerelated inflammation and oxidative stress in elderly macaque. Besides, the expression of inflammatory factors TNF-α, IL-1β, and IL-17A in the peripheral blood was analyzed after 30，60 and 90 days after BMMSC treatment. After 180 days histological and ROS level analysis was performed and the results showed that the serum IL-1β level was significantly reduced 30 days after BMMSC treatment, but the level returned to normal at 60 and 90 days. However, western blot analysis of the lung tissue, showed that IL-1β remained relatively lower than in the model group after 6 months of treatment.
Furthermore, TNF-α expression levels appeared to be inconsistent in the lungs and peripheral blood. The content of TNF-α in the peripheral blood decreased significantly after 30 days of cell transfusion and returned to the levels before treatment at 60 and 90 days. However, after 6 months, western blot analysis of the lung tissue, found that TNFα expression levels were significantly lower than in the model group. This inconsistency between the circulation and lung tissue expression levels differed from that reported in previous studies on rodent lung injury models [24,25] . Besides, our results show that IL-6 was downregulated in the lung tissue compared to the model group after significantly reduced [26] . IL-10 is a multi-functional cytokine, which regulates the growth and differentiation of cells, participates in inflammatory reactions and immune responses, and is recognized as an immunomodulatory cytokine used by almost all innate immune cells [27] . Current studies have shown that the number and function of Treg cells have a regulatory effect on CD4 + and CD25-cells to activate the release of IL-10. Aging affects the ability of CD4 (+), CD25 (+), FOXP3 (+) T cells to regulate the production of IL-10 [28] . Besides, mesenchymal stem cells, are powerful immune regulatory cells, with not only a regulatory effect on Treg cells but also strongly influence IL-10 production. Some studies have reported that mesenchymal stem cells can increase the production of IL-10 by communicating with macrophages, B lymphocytes, and dendritic cells or by secreting PGE2, ID0, IL-6, and HO-1 [29] . In this study, CD4, CD25, and FOXP3 were used as markers to detect the changes in the ratio of Treg cells in peripheral blood within 90 days after mesenchymal stem cell treatment.
Treg cells in peripheral blood were found to continuously decrease from 0 to 60 days after BMMSC treatment, and the low levels were maintained from 60 to 90 days. These results were inconsistent with those reported in previous studies on the regulation of Treg cells by mesenchymal stem cells under extremely high inflammatory conditions [29] . Therefore, it was not clear whether BMMSC regulated the entry of Treg cells into the tissues, hence reducing the Treg ratio in peripheral blood. Therefore, FOXP3 was used as a marker to further explore the level of Treg cells in the lung tissue. However, the findings from the lung tissues were consistent with those in the peripheral blood, but the FOXP3 levels in the lung tissue of the treatment group were significantly reduced. Therefore, this was speculated to be related to the extremely high inflammation levels, and the mesenchymal stem cells are not sufficient enough to adjust the inflammation level to a normal state and thus requires more Treg cells to be mobilized to deal with the extremely high inflammation. However, under normal aging conditions, the body's inflammation is not very high and can be well controlled by mesenchymal stem cells, which may not require too many Treg cells to be mobilized.
This therefore, leads to reduced Treg levels. The content of IL-10 in the lung tissue was detected by western blot analysis. The results showed that after 6 months of BMMSC treatment, the content of IL-10 in the lung tissue of macaque was significantly higher than that of the model group. These findings were consistent with the short-term observation in a previous mesenchymal stem cell treatment model of lung injury [30] .
However, the decrease in the ratio of Treg cells was inconsistent with the phenomenon of elevated IL-10, and this shows that BMMSC treatment can promote the secretion of IL-10 through other mechanisms not related to Treg.
In summary, this study reveals for the first time that BMMSC delays aging-related lung degeneration in the elderly macaque. This study had limitations. Due to experimental limitations, the impact of BMMSC on lung function was not explored in this study. Besides, the mechanisms by which BMMSC improved lung degeneration was also not explored.

Conclusions
1.Obtaining elderly model of pulmonary degenerative macaque shows that the alveolar cavity is enlarged, the structure of the lung is disordered, the pigmentation is increased, the degree of fibrosis is increased, and the capillary density is decreased.
2. BMMSC can reduce the degree of pulmonary fibrosis in elderly macaques, reduce the level of inflammation in the lung and peripheral blood, increase the expression of VEGF in lung tissue, increase the density of capillaries around the alveoli,and reduce the content of Treg cells in peripheral blood and lung tissue.
3. BMMSC can reduce the expression of type Ⅱ alveolar epithelial aging-related genes, reduce its apoptosis and oxidative stress levels, and promote proliferation.
BMMSC can increase the number of type Ⅱ alveolar epithelium in lung tissue.

Declarations
• Ethics approval and consent to participate Experimental protocols were approved by the Experimental Animal Ethics Committee of the 920th Hospital of the PLA Joint Logistics Support Force.

• Consent for publication
Not applicable.

• Availability of data and material
All data generated or analysed during this study are included in this published article.

• Competing interests
The authors declare that they have no competing interests.

Authors' contributions
YKY,XQZ,YLand YYW made substantial contributions to study conception and design, data acquisition, or data analysis and interpretation.
YKY and RGP agree to be accountable for all aspects of the work and ensure that questions related to the accuracy or integrity of any part of the work will be appropriately investigated and resolved.
XHP and XQZ have given final approval of this version of the manuscript for publication.
XQZ, XHP, HYH, YKY and RQP have been involved in drafting the manuscript or revising it critically for important intellectual content.