Changes in Elastic Fibres in Yak Lungs at Different Developmental Stages

Yaks have a strong adaptability to the plateau environment, which is closely associated with the effective oxygen utilization rate of their lung tissue. The elastic bre is an important adaptive structure of alveolar tissue. However, there are few studies on the development of the structure of lung tissue and the changes in elastic bres of yak after birth. The purpose of this study was to investigate the changes of elastic bers in the lungs of yaks after birth and the relationship between these changes and adaptation to hypoxic environment.


Abstract Background
Yaks have a strong adaptability to the plateau environment, which is closely associated with the effective oxygen utilization rate of their lung tissue. The elastic bre is an important adaptive structure of alveolar tissue. However, there are few studies on the development of the structure of lung tissue and the changes in elastic bres of yak after birth. The purpose of this study was to investigate the changes of elastic bers in the lungs of yaks after birth and the relationship between these changes and adaptation to hypoxic environment.

Results
In this experiment, a histological method was employed to observe the changes in the lung tissue structure of yaks at four ages: 1 day old, 30 days old, 180 days old and adult. There was no signi cant difference in the area of a single alveolus between the 1-day-old and 30-day-old groups (P > 0.05). In yaks aged over 30 days, the single alveolar area gradually increased with age (P < 0.05). The observation of elastic bres showed that elastic bres in alveolar tissue increased signi cantly from the ages of 30 days to 180 days (P < 0.05) and stabilized after 180 days of age. Transcriptome analysis determined the highest levels of differentially expressed genes between 30 days of age and 180 days of age. KEGG analysis showed that the PI3K-Akt signalling pathway and MAPK pathway, which are involved in bre formation, accounted for the largest proportion of differentially expressed genes between 30 days of age and 180 days of age. The expression levels of 36 genes related to bre formation were analysed, and several genes related to elastic bre formation and collagen bre formation were determined to be highly expressed at the age of 30 days.

Conclusions
The content of elastic bres in the alveolar tissue of yaks increases signi cantly after birth, but this change occurs only from 30 days of age to 180 days of age to make better use of oxygen in the environment.

Background
Yak is the only bovine animal that can multiply in the arctic-alpine pastoral area of the Qinghai-Tibet Plateau. This animal has strong adaptability to its ecological environment, and it is hard-working. Yak can live freely and multiply under harsh environmental conditions, such as hypoxia, cold and short herbage growing periods. Yak is important to plateau animal husbandry and is an essential means of livelihood and production for local people, being well-known as the "ship of the plateau" and "all-round livestock" [1]. With the constant development of yak resources, it can be stated that the sustainable development of the yak economy has become the highest priority of plateau animal husbandry.
Yak is a kind of animal with good adaptability in plateau environments, developing and reproducing in a special and harsh eco-geographical environment. After a long period of natural and arti cial selection, yak has developed characteristics of morphology, physiology and heredity that are different from those of other animals [2]. Yak has already attracted wide attention from scholars, both in this country and abroad, due to its good adaptability to plateau environments. At present, there are many studies on the morphological structure of organs and tissues in adult yaks [3]. Anatomically, yak ribs are relatively long, and the intercostal spacing is large, which can increase the chest size and provide a useful space for the development of the heart and lungs. Also, the large diameter of the yak windpipe can increase the amount of air entering the body [4]. In microanatomy, a respiratory system histology study found that the yak trachea is rich in goblet cells, the alveolar diaphragm is thick, the pulmonary arterioles are thin, and the gas-blood barrier is relatively thin, which is conducive to the passage and diffusion of oxygen [5]. In the yak cardiovascular system, enhancing the conduction of excitement by increasing conduction bres increases the length and density of capillaries in the heart, thereby increasing oxygen delivery [6].
Regarding skeletal muscle histology, yak muscle bre diameter is relatively small, which increases the density of muscle bre per unit area. In addition, the elastic bre content in yak muscle bre is relatively rich, which effectively improves its adaptability to hypoxia [7]. Physiologically, yak's red blood cell number and haemoglobin content are relatively high, and these values increase with increasing altitude, which helps to enhance yak's ability to carry oxygen in the blood [8]. However, there are few studies on the development of the structure of lung tissue and the changes in elastic bres of yak after birth.
The elastic bres have many branches and wide distribution, being interwoven into a net and arranged into a lm in lung tissue. Elastic membranes are alternately combined to form elastic membrane units, that is, elastic arterial resilience units [9,10]. According to previously published research, ripe elastic bres and elastic membranes are composed of homologous elastin macromolecules, which form scaffolds along micro brils arranged in parallel [11,12]. Because of the elastic bres, which are bene cial to gas exchange, the lungs have good elasticity [13]. Therefore, we investigated the changes in elastic bres in yak lung tissues at different developmental stages from the perspective of histological observation. The mechanism governing formation was further elucidated by molecular biological detection to provide basic ndings on the histological and molecular mechanisms underlying yak adaptation to hypoxia and to establish a foundation for future research in plateau medicine and other disciplines.

Results
Observation of the basic structure of yak alveolar tissues at different developmental stages It was observed that the alveolar morphology of yak was similar at different ages, and most of the alveoli were irregular oblate and oval. One-day-old yaks had different alveolar sizes, which were smaller than those of 30-day-old, 180-day-old and adult yaks. Elastic bres were determined to be evenly distributed in the alveolar septum, while elastic bres at the top of the alveolar septum were obviously distributed. Some translucent structures could be observed in the alveoli of 180-day-old adults, and the number of elastic bres increased signi cantly (Fig. 1A). According to quantitative analysis, the number of alveoli per unit area was not signi cantly different between the 1-day-old and 30-day-old groups (P > 0.05) but decreased signi cantly between the 30-day-old group and the adult group (P < 0.05) (Fig. 1B). The average single alveolar area exhibited no signi cant difference between the 1-day-old group and the 30day-old group (P > 0.05), but it gradually increased signi cantly from the 30-day-old group to the adult group (P<0.05) (Fig. 1C). The percentage of elastic bres in the lung parenchyma showed an increasing trend, but there was no signi cant difference between the 1-day-old group and the 30-day-old group (P > 0.05), and it increased signi cantly from the 30-day-old group to the 180-day-old group (P < 0.05), and there was no signi cant difference between the 180-day-old group and the adult group (P > 0.05) (Fig.   1D).

GO analysis of differentially expressed genes
Through GO enrichment analysis (Fig. 3), the differentially expressed genes of 1-day-old yaks vs. 30-dayold yaks, 30-day-old yaks vs. 180-day-old yaks, and 180-day-old yaks vs. adult yaks were analysed. The main biological processes (BP) involved in differential gene expression in each group include developmental processes and stimulus stress. Cell composition (CC) mainly concentrates action on several membrane and intimal systems. The molecular function (MF) mainly involves the functions of protein binding and ion binding.

KEGG analysis
The differentially expressed genes screened in each group were analysed by KEGG to determine the main biochemical metabolic pathways and signal transduction pathways involved in differentially expressed genes. Analysis found that the PI3K-Akt signalling pathway was mapped in the differentially expressed genes between two adjacent ages (Fig. 4). Among the various pathways, the PI3K-Akt signalling pathway is the most important pathway involved in differentially expressed genes observed from 30 days of age to 180 days of age, and the second most important pathway is the MAPK signalling pathway (Fig. 4B).

Screening of Fibrogenic Genes
By reviewing relevant studies conducted both in this country and abroad and combining them with GO annotation, we identi ed 36 genes involved in bre production ( Table 2). Among these genes, 22 were upregulated (61.11%), and 14 were downregulated (38.89%). Two genes were differentially expressed between 1-day-old and 30-day-old yaks, and 34 genes were differentially expressed between 30-day-old and 180-day-old yaks. There was no signi cant difference in the expression of these genes between 180day-old and adult yaks.
Expression trend of brogenesis-related genes in different periods Five genes related to elastic bre formation were selected, and all ve genes promoted bre formation.
The expression levels of yak lung tissue in different stages was analysed, and it was found that the expression level was the highest at 30 days of age or 180 days of age (Fig. 5A). Seven genes related to broblasts were selected, and their functions may also promote brogenesis. The expression level was the highest in the 30-day-old and 180-day-old groups (Fig. 5B). Moreover, 24 genes related to collagen bre formation were selected, among which 20 genes promoted bre formation. The expression levels of 9 genes were the highest at 30 days of age (Fig. 5C), and 11 genes were the highest at 180 days of age (Fig. 5D). Then, 4 genes had inhibitory effects on brogenesis, and their expression levels all decreased continuously after 30 days of age (Fig. 5E).

Discussion
Observation of yak alveolar tissue slices at different developmental stages The alveolus is the functional unit of the lungs, and gas exchange in the lungs largely depends on the size of the respiratory area of the lungs [14]. With the growth and development of the yak, individual volume and surface area increase, and the number of alveoli per unit area decrease (Fig. 1B). The total number of alveoli increases, which increases the area of gas exchange in the lung, accelerates the rate of gas exchange in lung tissue [15], and improves the utilization rate of oxygen, enabling yaks to adapt quickly to low-oxygen and high-altitude environments. The ability of yaks to adapt to low oxygen at high altitudes has representative signi cance [16].
The elastic bres have a retractive force in alveolar tissue, and the adult yaks tend to ripen; therefore, it is more bene cial for yaks to exchange air between the outside atmosphere and the blood in the lungs by using their own retraction force. This property helps blood vessels bear the pressure of the heartbeat and blood ow to keep the blood ow constant [17]. The proportion of elastic bres in yak alveoli was observed to increase signi cantly after 30 days of age (P < 0.05) (Fig. 1D). Therefore, it can be seen that 30 days of age is the key period of yak alveolar development. The alveolar tissues of 180-day-old adults exhibit some ribbon alveolar septum structure of the semipermeable membrane, and the related literature presents similar reports [18]. The location of the elastic bre that we observed was consistent with this translucent membrane structure, and it was inferred that it may be an elastic bre.
Expression analysis of differentially expressed genes By distinguishing the biological information of transcriptomic data between two age groups, it was found that the comparison of the 30-day-old group with the 180-day-old group yielded the highest level of differentially expressed genes (Fig. 2B). Consequently, the stage from 30 days old to 180 days old was indicated to involve many gene expression changes, as this period is a signi cant stage of yak lung tissue development. This result was consistent with our previous morphological observations.

GO and KEGG annotations
GO enrichment analysis showed that the biological process of differentially expressed genes in three age groups involved the development process, mainly in the membrane and nucleus. The main biological processes were biological regulation and metabolism, and the process involved the functions of protein binding and ion binding. The proportion of differentially expressed genes in the yak lung tissue was highest between the 30-day-old group and the 180-day-old group (Fig. 3B), which suggested that yak lung tissue underwent sustained development from 30 days of age to 180 days of age.
KEGG pathway analysis showed that the PI3K-Akt signalling pathway was an important cellular regulation pathway in three age groups and was related to the formation of bres [19]. The signalling pathways involved in the formation of bres were mostly observed between 30 days of age and 180 days of age [20]. Among these pathways, the PI3K-Akt signalling pathway accounted for the largest proportion ( Fig. 4B) followed by MAPK, which was also closely related to cell growth and development [21].

Genes related to brogenesis
Thirty-four of the 36 genes involved in bre formation were differentially expressed from 30 days of age to 180 days of age. It was found that a large number of bres were formed in this stage. Moreover, the expression levels of the genes related to promoting brogenesis increased signi cantly between 30 days of age and 180 days of age (P < 0.05), while the expression levels of the genes related to inhibiting brogenesis decreased at this stage. The genes related to elastic brogenesis are FBN1, FBN2, EMILIN3, EMILIN2 and ELN; FBN1 and FBN2 belong to the brillin protein family [22]; and EMILIN3, EMILIN2 and ELN belong to the elastin family. The brillin family and the elastin family genes are closely related to the formation of elastic bres [23]; therefore, we selected the ELN for analysis, and the expression level of ELN reached its maximum at 30 days of age (Fig. 5A); also, ELN was abundant in lung tissue. Elastin constitutes elastic bre, and natural elastin is surrounded by a shell composed of micro brils. Micro brils are composed of some glycoproteins, and brillin is necessary to maintain the integrity of elastic bres [24]. Elastic bre is a stretched rubber-like bre that can provide elasticity and tensile strength to tissues.
Although collagen can provide strength and toughness to the extracellular matrix, it needs to be elastic for lung tissue, and the elasticity primarily depends on elastic bres in the extracellular matrix.
There are 7 broblast-related genes, FGF1, FGF9, FGF18, FIBP, CNPY3, TLR3 and FN1, which can promote the formation of broblasts. Fibroblast growth factors have a wide range of biological activities that are closely related to cell proliferation and differentiation [25]. These factors can promote the mitosis of broblasts and the growth of mesodermal cells, stimulate the formation of blood vessels and play a role in wound healing and limb regeneration [26]. Fibroblast-related genes can promote the growth of broblasts and subsequently cause them to develop into broblasts [27]. The expression levels of related genes reached a maximum at 30 or 180 days of age (Fig. 5B), which shows that bre formation is upregulated at this stage.
There are 20 genes that promote the formation of collagen bres. The collagen family is primarily associated with cell composition, and other related genes mainly participate in bre formation by inducing related growth factors and various cytokines [28]. COL3A1 gene expression reached the highest value at 30 days of age (Fig. 5C). Type III collagen is a kind of high-molecular-weight protein. Filamentous collagen bres are the bonding materials of connective tissue, which can keep the skin rm and elastic, participate in the migration, differentiation and proliferation of cells, and promote the generation of collagen bres [29]. Glutathione peroxidase can improve the survival rate of cells and ensure the integrity of genetic DNA [30]. The GPX1 gene reached its maximum expression level at 180 days of age (Fig. 5D), con rming that GPX can promote collagen bre formation [31]. Four genes, ADAMTS2, ACAN, TGFβ2 and TGFβ1, repress the formation of collagen bres. These genes are mainly involved in inducing (Fig. 5E) and inhibiting the effects of growth factors and cytokines to inhibit bre formation.

Conclusions
In conclusion, this study proved that yak's good adaptability to plateau hypoxia environment was closely related to the elastic bers in alveolar tissue. For example, during the development process, the differentially expressed genes were at the highest level from 30 days old to 180 days old, PI3K-Akt signaling pathway and MAPK pathway involved in ber formation accounted for the largest proportion, and genes related to ber formation were also highly expressed at 30 days old or 180 days old. To make better use of oxygen in the environment, the elastic bers in the alveolar tissue of yaks increased signi cantly from 30 to 180 days of age, and stabilized after 180 days. The existence of elastic bers makes the lungs have good elasticity, is conducive to gas exchange, improves the oxygen utilization rate of lung tissues, and enables yaks to better adapt to the environment.

Experimental animals
From the Haiyan area, Qinghai province of China (3200 m above sea level), there two 1-day-old and 180day-old plateau yaks were studied, as well as three 30-day-old and adult plateau yaks. All yaks purchased from herders of Haiyan area. The respiratory systems of these yaks were healthy, regardless of sex. All plateau yaks were killed by exsanguinated via abdominal aorta in slaughter house after anesthesia via IV injection of pentobarbital sodium (200 mg/kg) according to the Animal Ethics Procedures and Guidelines of the People's Republic of China.
Histological staining Para n sections of conventional tissues Fresh tissues were collected, xed with 4% paraformaldehyde for 24 hours, dehydrated with gradient alcohol, cleared with xylene, embedded in para n after wax immersion, sliced with a slicer to a thickness of 4 μm, and placed on glass slides for later use.

HE staining
For HE staining of tissue samples, reverse gradient alcohol rehydration was employed followed by staining with haematoxylin for 5 min, differentiating by diluted hydrochloric acid differentiation, rinsing fully in running water, treating tissues with 0.6% ammonia water until they turned blue, rinsing the tissues with running water, staining with eosin for 1-3 min, and sealing through gradient alcohol dehydration.
Elastic bre staining Tissue samples were subjected to reverse gradient alcohol rehydration with Wiegert oxidant for oxidation for 5 min, washed with Wiegert bleach for 1~2 min, differentiated with acidic differentiation solution for 2~3 s, washed with running water for 10 min, re-dyed with VG staining solution for 30 s, and nally sealed through gradient alcohol dehydration.
Observation and measurement HE-stained sections and elastic bre sections were observed with an Olympus BX51 microscope, and pictures were collected. Next, images were taken and measured with Image-Pro Plus 6.0, which was employed to measure the area of single alveoli and the number of alveoli per unit area in HE-stained sections; the areas of lung parenchyma and elastic bres in different developmental stages were also measured. Excel was used to calculate the proportion of elastic bres in alveolar tissue, and SPSS 19.0 was used to perform statistical analysis among multiple groups. The results are expressed as the mean ± standard deviation (x ̅ ± SD), with a P-value < 0.05 indicating a signi cant difference.

Transcriptome data analysis
Transcriptome sequencing Transcriptome sequencing was performed on the total RNA samples of yak lung tissue samples from 1day-old, 30-day-old, 180-day-old, and adult yaks by Shanghai Liebing Biomedical Technology Co., Ltd. Sequencing was performed the NovaSeq sequencing platform by adopting double-end sequencing mode, carrying out quality control (QC) and pollution assessment on the sequencing data, and then analysing the expression of the genes after quality control.
Bioinformatic analysis GO (gene ontology) databases and KEGG (Kyoto Encyclopedia of Genes and Genomes) databases were used to analyse transcriptomic data combined with veri ed transcriptomic data. For screening differentially expressed genes, the parameters are 1.5 times the difference and FDR (false discovery rate) ≤ 0.05. Screening and counting bre generation-related genes of samples in different periods was performed. The change trend of brogenic gene expression in each period was measured and analysed.  ), and all methods were carried out in accordance with approved guidelines. No local regulations or laws were overlooked. I had obtained written informed consent to use the animals in this study from the owners of the animals.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.   Table 2 Annotation of genes related to ber formation