Pig Lung Tissue Research Articles

Phenotypic and Genotypic Analysis of Antimicrobial Resistance in Mycoplasma hyopneumoniae Isolated from Pigs with Enzootic Pneumonia in Australia.

Mycoplasma hyopneumoniae, an important cause of enzootic pneumonia in pigs in many countries, has recently been shown to exhibit reduced susceptibility to several antimicrobial classes. In the present study, a total of 185 pig lung tissue samples were collected from abattoirs in Australia, from which 21 isolates of M. hyopneumoniae were obtained. The antimicrobial resistance profile of the isolates was determined for 12 antimicrobials using minimum inhibitory concentration (MIC) testing, and a subset (n = 14) underwent whole-genome sequence analysis. MIC testing revealed uniformly low values for enrofloxacin (≤1 μg/mL), florfenicol (≤8 μg/mL), lincomycin (≤4 μg/mL), spectinomycin (≤4 μg/mL), tetracycline (≤0.5 μg/mL), tiamulin (≤2 μg/mL), tildipirosin (≤4 μg/mL), tilmicosin (≤16 μg/mL) tulathromycin (≤2 μg/mL), and tylosin (≤2 μg/mL). Higher MICs were observed for erythromycin (MIC range: 16-32 μg/mL), gamithromycin, and tilmicosin (MIC range of both: 32-64 μg/mL). Whole-genome sequencing of the isolates and additional screening using mismatch amplification mutation assay PCR did not identify any known genetic resistance markers within 23S rRNA (macrolides), DNA gyrase A, and topoisomerase IV genes (fluoroquinolones). The WGS data also indicated that the Australian M. hyopneumoniae isolates exhibited limited genetic diversity and formed a distinct monophylectic clade when compared to isolates from other countries. These findings indicate that Australian M. hyopneumoniae likely remains susceptible to the major antimicrobials used to treat enzootic pneumonia in pigs and have evolved in isolation from strains identified in other pig-producing countries.

Ssc-miR-101-3p inhibits hypoxia-induced apoptosis and inflammatory response in alveolar type-II epithelial cells of Tibetan pigs via targeting FOXO3

Tibetan pigs are a unique swine strain adapted to the hypoxic environment of the plateau regions in China. The unique mechanisms underlying the adaption by Tibetan pigs, however, are still elusive. Only few studies have investigated hypoxia-associated molecular regulation in the lung tissues of animals living in the plateau region of China. Our previous study reported that ssc-miR-101-3p expression significantly differed in the lung tissues of Tibetan pigs at different altitudes, suggesting that ssc-miR-101-3p plays an important role in the adaptation of Tibetan pigs to high altitude. To understand the underlying molecular mechanism, in this study, the target genes of ssc-miR-101-3p and their functions were analyzed via various methods including qRT-PCR and GO and KEGG pathway enrichment analyses. The action of ssc-miR-101-3p was investigated by culturing alveolar type-II epithelial cells (ATII) of Tibetan pigs under hypoxic conditions and transfecting ATII cells with vectors overexpressing or inhibiting ssc-miR-101-3p. Overexpression of ssc-miR-101-3p significantly increased the proliferation of ATII cells and decreased the expression of inflammatory and apoptotic factors. The target genes of ssc-miR-101-3p were significantly enriched in FOXO and PI3K-AKT signaling pathways required to mitigate lung injury. Further, FOXO3 was identified as a direct target of ssc-miR-101-3p. Interestingly, ssc-miR-101-3p overexpression reversed the damaging effect of FOXO3 in the ATII cells. In conclusion, ssc-miR-101-3p targeting FOXO3 could inhibit hypoxia-induced apoptosis and inflammatory response in ATII cells of Tibetan pigs. These results provided new insights into the molecular mechanisms elucidating the response of lung tissues of Tibetan pigs to hypoxic stress.

New insight on porcine carboxylesterases expression and activity in lung tissues

Over the course of the last twenty years, there has been a growing recognition of the pig's potential as a valuable model for studying human drug metabolism. This study aimed to investigate the expression, enzymatic activity, inhibitory susceptibility, and cellular localization of carboxylesterases (CES) in porcine lung tissue not yet explored. Our results showed that CESs hydrolysis activity followed Michaelis-Menten kinetics in both cytosolic and microsomal fractions of porcine lung tissues (N = 8), with comparable hydrolysis rates for tested substrates, namely 4-nitrophenyl acetate (pNPA), 4-methylumbelliferyl acetate (4-MUA), and fluorescein diacetate (FD). We also determined the CESs hydrolysis activity in a representative sample of the porcine liver that, as expected, displayed higher activity than the lung ones. The study demonstrated variable levels of enzyme activities and interindividual variability in both porcine lung fractions. Inhibition studies used to assess the CESs' involvement in the hydrolysis of pNPA, 4-MUA, and FD suggested that CESs may be the enzymes primarily involved in the metabolism of ester compounds in the pig lung tissue. Overall, this study provides insight into the distribution and diversity of CES isoforms involved in substrate hydrolysis across different cellular fractions (cytosol and microsomes) in porcine lungs.

Ascaris suum infection in juvenile pigs elicits a local Th2 response in a setting of ongoing Th1 expansion.

Ascaris spp. undergo extensive migration within the body before establishing patent infections in the small intestinal tract of humans and pigs. However, whether larval migration is critical for inducing efficient type 2 responses remains poorly understood. Therefore, we investigated systemic versus local adaptive immune responses along the hepato-tracheal migration of Ascaris suum during primary, single infections in conventionally raised pigs. Neither the initial invasion of gut tissue nor migration through the liver resulted in discernable Th2 cell responses. In contrast, lung-stage larvae elicited a Th2-biased pulmonary response, which declined after the larvae had left the lungs. In the small intestine, we observed an accumulation of Th2 cells upon the arrival of fourth-stage larvae (L4) to the small intestinal lumen. In parallel, we noticed robust and increasing Th1 responses in circulation, migration-affected organs, and draining lymph nodes. Phenotypic analysis of CD4+ T cells specifically recognizing A. suum antigens in the circulation and lung tissue of infected pigs confirmed that the majority of Ascaris-specific T cells produced IL-4 (Th2) and, to a much lesser extent, IL-4/IFN-g (Th2/1 hybrids) or IFN-g alone (Th1). These data demonstrate that lung-stage but not the early liver-stage larvae lead to a locally restricted Th2 response. Significant Th2 cell accumulation in the small intestine occurs only when L4 complete the body migration. In addition, Th2 immunity seems to be hampered by the concurrent, nonspecific Th1 bias in growing pigs. Together, the late onset of Th2 immunity at the site of infection and the Th1-biased systemic immunity likely enable the establishment of intestinal infections by sufficientlylarge L4 stages and pre-adult worms, some of which resist expulsion mechanisms.

Feasibility of Near-Infrared Spectroscopy in the Classification of Pig Lung Lesions.

Respiratory diseases significantly affect intensive pig farming, causing production losses and increased antimicrobial use. Accurate classification of lung lesions is crucial for effective diagnostics and disease management. The integration of non-destructive and rapid techniques would be beneficial to enhance overall efficiency in addressing these challenges. This study investigates the potential of near-infrared (NIR) spectroscopy in classifying pig lung tissues. The NIR spectra (908-1676 nm) of 101 lungs from weaned pigs were analyzed using a portable instrument and subjected to multivariate analysis. Two distinct discriminant models were developed to differentiate normal (N), congested (C), and pathological (P) lung tissues, as well as catarrhal bronchopneumonia (CBP), fibrinous pleuropneumonia (FPP), and interstitial pneumonia (IP) patterns. Overall, the model tailored for discriminating among pathological lesions demonstrated superior classification performances. Major challenges arose in categorizing C lungs, which exhibited a misclassification rate of 30% with N and P tissues, and FPP samples, with 30% incorrectly recognized as CBP samples. Conversely, IP and CBP lungs were all identified with accuracy, precision, and sensitivity higher than 90%. In conclusion, this study provides a promising proof of concept for using NIR spectroscopy to recognize and categorize pig lungs with different pathological lesions, offering prospects for efficient diagnostic strategies.

Exploring the role of the CapG gene in hypoxia adaptation in Tibetan pigs.

Introduction: The CapG gene, which is an actin-binding protein, is prevalent in eukaryotic cells and is abundantly present in various pathways associated with plateau hypoxia adaptation. Tibetan pigs, which have inhabited high altitudes for extended periods, provide an excellent research population for investigating plateau hypoxia adaptation. Results: This study focused on Tibetan pigs and Yorkshire pigs residing in Nyingchi, Tibet. The blood physiological data of Tibetan pigs were found to be significantly higher than those of Yorkshire pigs, including RBC, HGB, HCT, MCH, and MCHC. The SNP analysis of the CapG gene identified six sites with mutations only present in Tibetan pigs. Notably, the transcription factors at sites C-489T, C-274T, and A-212G were found to be altered, and these sites are known to be associated with hypoxia adaptation and blood oxygen transportation. The mRNA expression of the CapG gene exhibited highly significant differences in several tissues, with the target proteins predominantly higher in the Yorkshire pig compared to the Tibetan pig. Specifically, a notable difference was observed in the lung tissues. Immunohistochemistry analysis revealed high expression levels of CapG proteins in the lung tissues of both Tibetan and Yorkshire pigs, primarily localized in the cytoplasm and cell membrane. Conclusion: The CapG gene plays a significant role in regulating hypoxia adaptation in Tibetan pigs. This study provides a theoretical basis for the conservation and utilization of Tibetan pig resources, the breeding of highland breeds, epidemic prevention and control, and holds great importance for the development of the highland livestock economy.

MiR-339-5p inhibits replication of porcine reproductive and respiratory syndrome virus by targeting viral gene regions.

Porcine reproductive and respiratory syndrome virus (PRRSV) is a variable virus, whose spread cannot be totally stopped by vaccination. PRRSV infection results in abortion and respiratory symptoms in pregnant pigs. One crucial component of the anti-viral infection strategy is microRNA (miRNA), a class of multifunctional small molecules. It is unknown whether miR-339-5p can specifically target the PRRSV gene and prevent the virus from replicating, despite the fact that miR-339-5p is markedly up-regulated during the PRRSV infection. In this pursuit, the present study revealed that the two PRRSV areas targeted by miR-339-5p were PRRSV nsp2-3378 to 3403 and PRRSV nsp2-3112 to 3133 using the miRanda program. Dual luciferase reporter assays showed that the miR-339-5p target region of the PRRSV gene sequence exhibited 100% homology and was highly conserved. Furthermore, the ability of miR-339-5p to target PRRSV gene areas was verified. It was found that the overexpression of miR-339-5p markedly reduced the PRRSV replication through PRRSV infection trials. The precursor sequence of ssc-miR-339-5p was amplified using the DNA of pig lung tissue as a template in order to create a fragment of 402bp of porcine-derived miR-339-5p precursor sequence, which was then used to produce the eukaryotic expression plasmid of miR-339-5p. In conclusion, miR-339-5p can target the specific PRRSV gene areas and prevent PRRSV replication, offering fresh perspectives for the creation of medications that combat the PRRSV infection.

A Methodological Approach for Interpreting and Comparing the Viscoelastic Behaviors of Soft Biological Tissues and Hydrogels at the Cell-Length Scale

The behavior of a cell is strongly influenced by the physical properties and stimuli in its microenvironment. Furthermore, the activation and modulation of mechanotransduction pathways are involved in tissue development and homeostasis and even pathological processes. Thus, when developing materials aimed at mimicking the extracellular matrixes of healthy or pathological tissues, their mechanical features should be closely considered. In this context, nanoindentation represents a powerful technique for mechanically characterizing biological tissues and hydrogels at the cell-length scale. However, standardized experimental protocols and data analysis techniques are lacking. Here, we proposed a methodological approach based on the nanoindentation technique for quantitatively analyzing and comparing the time-dependent load relaxation responses of soft biological tissues and hydrogels. As this was an explanatory study, stress-relaxation nanoindentation tests were performed on samples of pig and human lung tissues and of a specific gelatin-methacryloyl (GelMA) hydrogel to quantify and compare their viscoelastic properties. The proposed method allowed for identifying the characteristic parameters needed for describing the behavior of each sample, permitting us to quantitatively compare their mechanical behaviors. All samples showed load relaxation at a defined indentation depth because of their intrinsic viscoelastic behaviors, and the GelMA samples showed the highest relaxation capabilities. The distribution of the characterization parameters showed that the biological samples presented similar time-dependent responses, while differences were observed in the GelMA samples. Overall, the proposed methodological approach allows for providing key insights into the time-dependent behaviors of soft biological tissues and hydrogels at the cell-length scale in view of supporting tissue engineering and pathophysiological investigations.

First Detection of Influenza D Virus Infection in Cattle and Pigs in the Republic of Korea.

Influenza D virus (IDV) belongs to the Orthomyxoviridae family, which also include the influenza A, B and C virus genera. IDV was first detected and isolated in 2011 in the United States from pigs with respiratory illness. IDV circulates in mammals, including pigs, cattle, camelids, horses and small ruminants. Despite the broad host range, cattle are thought to be the natural reservoir of IDV. This virus plays a role as a causative agent of the bovine respiratory disease complex (BRDC). IDV has been identified in North America, Europe, Asia and Africa. However, there has been no information on the presence of IDV in the Republic of Korea (ROK). In this study, we investigated the presence of viral RNA and seroprevalence to IDV among cattle and pigs in the ROK in 2022. Viral RNA was surveyed by the collection and testing of 999 cattle and 2391 pig nasal swabs and lung tissues using a real-time RT-PCR assay. IDV seroprevalence was investigated by testing 742 cattle and 1627 pig sera using a hemagglutination inhibition (HI) assay. The viral RNA positive rate was 1.4% in cattle, but no viral RNA was detected in pigs. Phylogenetic analysis of the hemagglutinin-esterase-fusion (HEF) gene was further conducted for a selection of samples. All sequences belonged to the D/Yamagata/2019 lineage. The seropositivity rates were 54.7% in cattle and 1.4% in pigs. The geometric mean of the antibody titer (GMT) was 68.3 in cattle and 48.5 in pigs. This is the first report on the detection of viral RNA and antibodies to IDV in the ROK.

Establishment of Streptococcus suis Biofilm Infection Model In Vivo and Comparative Analysis of Gene Expression Profiles between In Vivo and In Vitro Biofilms.

Streptococcus suis is a zoonotic pathogen that continuously threatens animal husbandry and public health worldwide. Studies have shown that S. suis can cause persistent infection by forming biofilms. In this study, a model of S. suis biofilm-related infection was successfully constructed for the first time by simulating the natural infection of S. suis, and biofilm of S. suis in vivo was successfully observed in the lung tissue of infected pigs by a variety of detection methods. Subsequently, selective capture of transcribed sequences (SCOTS) was used to identify genes expressed by S. suis in vivo biofilms. Sixty-nine genes were captured in in vivo biofilms formed by S. suis for the first time by SCOTS; they were mainly involved in metabolism, cell replication, and division, transport, signal transduction, cell wall, etc. Genes related to S. suis in vitro biofilm formation were also identified by SCOTS and RNA sequencing. Approximately half of the genes captured by SCOTS in the in vivo and in vitro biofilms were found to be different. In summary, our study provides powerful clues for future exploration of the mechanisms of S. suis biofilm formation. IMPORTANCE Streptococcus suis is considered an important zoonotic pathogen, and persistent infection caused by biofilm is currently considered to be the reason why S. suis is difficult to control in swine. However, to date, a model of the biofilm of S. suis in vivo has not been successfully constructed. Here, we successfully detected biofilms of S. suis in vivo in lung tissues of piglets infected with S. suis. Selective capture of transcribed sequences and the transcriptome were used to obtain gene profiles of S. suis in vivo and in vitro biofilms, and the results showed large differences between them. Such data are of importance for future experimental studies exploring the mechanism of biofilm formation by S. suis in vivo.

Detection of microplastics in domestic and fetal pigs’ lung tissue in natural environment: A preliminary study

Microplastics (MPs) are ubiquitous in the environment. However, it is unclear whether MPs are present in mammalian lungs through inhalation, and if so, could be possibly found in fetal tissues. In this study, we aim to determine the presence and characteristics of particles in domestic and fetal pig lung tissue in the natural environment. Specimens from the lungs of domestic pigs (n = 10) and fetal pigs that already died in matrix during vaginal birth from the non-contaminated area (n = 10) were obtained from farmers’ nearby sludge treatment plant. These specimens were compressed between two glass microscope slides, which were examined under polarized light microscopy. In addition, Agilent 8700 LDIR Chemical imaging system (LDIR) was used to determine the quantitative and qualitative characteristics of MPs. According to the polarized light microscope survey of domestic pig lungs, we observed an average of 12 particles/g, which was more than the 6 particles/g observed in fetal pig lungs, which ranged in size from 115.14 μm to 1370.43 μm. All the observed MP particles were fiber in shape. LDIR indicated an average of 180 particles/g of domestic pig lungs, ranging in size from 20.34 μm to 916.36 μm, which was twice as many MPs observed in fetal pig lungs. Furthermore, the compositions of MPs were different between them. LDIR indicated that polyamide (PA) was the most common polymer identified in domestic pig lungs (46.11%), while polycarbonate (PC) was the most common polymer in fetal pig lungs (32.99%). These findings confirmed the presence of MPs in the lung tissue of both domestic and fetal pigs in the natural environment, but the main characteristics differed. This fact indicated the increasing risk of MPs to human respiratory tract is increasing. Further research should be conducted to entirely estimate the specific exposure level on humans and offspring.

P34L Mutation of swine TIM-1 enhances its ability to mediate Japanese encephalitis virus infection

Japanese encephalitis virus (JEV) is a major causative agent of neurological infection affecting humans and pigs. Human T Cell Immunoglobulin and Mucin Domain 1 (hTIM-1) enhances the infection of JEV through virion-associated phosphatidylserine (PS) binding. Here, five swine TIM-1 (sTIM-1) gene variants were cloned from pig lung tissues by reverse-transcriptase polymerase chain reaction (RT-PCR). Sequence alignment analysis revealed that the gene homology between the sTIM-1 and hTIM-1 was 42.3–43.8%. Furthermore, ectopic expression of all five sTIM-1 variants in 293 T cells can promote JEV entry and infection. However, sTIM-1 V3 exhibited significantly less potent at promoting virus entry compared to the other four variants. Further studies revealed that the 34th amino acid of sTIM-1is critical for the entry of JEV, which is Pro34 in sTIM-1V3 while Leu34 in other four sTIM-1 variants. Mechanically, leucine at locus 34 was associated with the membrane distribution of sTIM-1, thereby affecting viral entry and infection. In total, our findings provide evidence that the PS receptor sTIM-1 promotes the infection of JEV and that the 34th amino acid position is critical for sTIM-1 to mediate viral infection.

Plateau Adaptation Gene Analyses Reveal Transcriptomic, Proteomic, and Dual Omics Expression in the Lung Tissues of Tibetan and Yorkshire Pigs.

Simple SummaryThe low oxygen concentrations of high-altitude regions hinder their development possibilities. In this investigation, we used lung tissue from the adopted Yorkshire sow and from the Tibetan pig to analyze the occurrence and development mechanisms of high-altitude hypoxia using dual expression omics. Seven key candidate genes (SELENBP1, MCC, CAPG, ASS1, ADH4, LYZ, and CPS1) were screened from the lung tissues and found to be predominately involved in mitochondrial function, blood particle regulation, glycolysis, ethanol oxidation, and the Wnt signaling pathway, as well as other related hypoxia-adaptive regulatory mechanisms.Elevated environments such as plateaus are often classified as low oxygen environments. The hypoxic adaptation mechanisms utilized by organisms in these conditions are not well understood. To address this, the differentially expressed genes (DEGs) involved in hypoxia adaptation were assessed using two pig breeds (Tibetan pig [TP] and Yorkshire sow [YY]). Genes related to lung tissue responses to hypoxia were assessed using transcriptomic (using RNA-seq) and proteomic (using iTRAQ) analysis. A total of 1021 DEGs were screened out. In the iTRAQ omics data, a total of 22,100 peptides were obtained and 4518 proteins were found after filtering. A total of 271 differentially expressed proteins [DEPs] were screened using the conditions of p < 0.05; FC ≤ 0.833; and FC ≥ 1.2. A total of 14 DEGs at the mRNA and protein levels were identified and found to be associated with regulation of the inflammatory response; blood particles; and MAPK cascade response regulation. Among the DEGs, six were associated with hypoxia adaptation function (mitochondria and glycolysis) in pigs. The results of this study identify novel candidate genes involved in porcine hypoxia adaptation mechanisms.

Raman-Guided Bronchoscopy: Feasibility and Detection Depth Studies Using Ex Vivo Lung Tissues and SERS Nanoparticle Tags

Image-guided and robotic bronchoscopy is currently under intense research and development for a broad range of clinical applications, especially for minimally invasive biopsy and surgery of peripheral pulmonary nodules or lesions that are frequently discovered by CT or MRI scans. Optical imaging and spectroscopic modalities at the near-infrared (NIR) window hold great promise for bronchoscopic navigation and guidance because of their high detection sensitivity and molecular/cellular specificity. However, light scattering and background interference are two major factors limiting the depth of tissue penetration of photons, and diseased lesions such as small tumors buried under the tissue surface often cannot be detected. Here we report the use of a miniaturized Raman device that is inserted into one of the bronchoscope channels for sensitive detection of “phantom” tumors using fresh pig lung tissues and surface-enhanced Raman scattering (SERS) nanoparticle tags. The ex vivo results demonstrate not only the feasibility of using Raman spectroscopy for endoscopic guidance, but also show that ultrabright SERS nanoparticles allow detection through a bronchial wall of 0.85 mm in thickness and a 5 mm-thick layer of lung tissue (approaching the fourth-generation airway). This work highlights the prospects and potential of Raman-guided bronchoscopy for minimally invasive imaging and detection of lung lesions.

Porcine β-defensin 2 confers enhanced resistance to swine flu infection in transgenic pigs and alleviates swine influenza virus-induced apoptosis possibly through interacting with host SLC25A4

Swine influenza virus (SIV) not only brings about great economic losses on the global pig industry, it also poses a significant threat to the public health for its interspecies transmission capacity. Porcine β-defensin 2 (PBD-2) is a host defense peptide and our previous study has shown that PBD-2 inhibits proliferation of enveloped pseudorabies virus both in vitro and in transgenic (TG) mice. The aim of this study is to investigate the possible anti-SIV ability of PBD-2 in a TG pig model created in our previous study. The in-contact challenge trial demonstrated that overexpression of PBD-2 in pigs could efficiently alleviate SIV-associated clinical signs. The SIV titers quantified by EID50 in lung tissues of infected TG pigs were significantly lower than that of wild-type littermates. In vitro, the cell viability assay revealed that PBD-2 mainly interfered with viral entry and post-infection stages. It was further confirmed that PBD-2 could enter porcine tracheal epithelial cells. The proteins interacting with PBD-2 inside host cells were identified with immunoprecipitation and the pathways involved were analyzed. Results showed that PBD-2 could interact with pro-apoptotic solute carrier family 25 member 4 (SLC25A4), also known as adenine nucleotide translocase 1, and thereby inhibited SIV-induced cell apoptosis. The molecular docking analysis suggested that PBD-2 interacted with porcine SLC25A4 mainly through strong hydrogen binding, with the predicted binding affinity being −13.23 kcal/mol. Altogether, these indicate that PBD-2 protects pigs against SIV infection, which may result from its role as a SLC25A4 blocker to alleviate cell apoptosis, providing a novel therapeutic and prophylactic strategy of using PBD-2 to combat SIV.

In Situ Hybridization of PRRSV-1 Combined with Digital Image Analysis in Lung Tissues of Pigs Challenged with PRRSV-1.

Betaarterivirus suid 1 and 2 are the causative agents of porcine reproductive and respiratory syndrome (PRRS), which is one of the most significant diseases of the swine industry, causing significant economic losses in the main pig producing countries. Here, we report the development of a novel, RNA-based in situ hybridization technique (RNAscope) to detect PRRS virus (PRRSV) RNA in lung tissues of experimentally infected animals. The technique was applied to lung tissues of 20 piglets, which had been inoculated with a wild-type, highly pathogenic PRRSV-1 strain. To determine the RNAscope’s applicability as a semi-quantitative method, we analysed the association between the proportion of the virus-infected cells measured with an image analysis software (QuPath) and the outcome of the real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) tests performed in parallel. The results of the quantitative approach of these two molecular biological methods show significant association (pseudo R2 = 0.3894, p = 0.004). This is the first time RNAscope assay has been implemented for the detection of PRRSV-1 in experimental animals.

The Expression Regulatory Network in the Lung Tissue of Tibetan Pigs Provides Insight Into Hypoxia-Sensitive Pathways in High-Altitude Hypoxia.

To adapt to a low-oxygen environment, Tibetan pigs have developed a series of unique characteristics and can transport oxygen more effectively; however, the regulation of the associated processes in high-altitude animals remains elusive. We performed mRNA-seq and miRNA-seq, and we constructed coexpression regulatory networks of the lung tissues of Tibetan and Landrace pigs. HBB, AGT, COL1A2, and EPHX1 were identified as major regulators of hypoxia-induced genes that regulate blood pressure and circulation, and they were enriched in pathways related to signal transduction and angiogenesis, such as HIF-1, PI3K-Akt, mTOR, and AMPK. HBB may promote the combination of hemoglobin and oxygen as well as angiogenesis for high-altitude adaptation in Tibetan pigs. The expression of MMP2 showed a similar tendency of alveolar septum thickness among the four groups. These results indicated that MMP2 activity may lead to widening of the alveolar wall and septum, alveolar structure damage, and collapse of alveolar space with remarkable fibrosis. These findings provide a perspective on hypoxia-adaptive genes in the lungs in addition to insights into potential candidate genes in Tibetan pigs for further research in the field of high-altitude adaptation.

Pig lung fibrosis is active in the subacute CdCl2 exposure model and exerts cumulative toxicity through the M1/M2 imbalance

Environmental pollutant cadmium (Cd) can cause macrophage dysfunction, and the imbalance of M1/M2 is involved in the process of tissue fibrosis. In order to explore the effect of subacute CdCl2 exposure on pig lung tissue fibers and its mechanism, based on the establishment of this model, ICP-MS, H&E staining, Masson staining, Immunofluorescence, RT-PCR, and Western Blot methods were used to detect related indicators. The results found that lung tissue fibrosis, Cd content significantly increased, lung tissue ion disturbance, miR-20a-3p down-regulation, M1/M2 imbalance, LXA4/FPR2 content decreased, MDA content increased, NF-κB/NLRP3, TGFβ pathway, PPARγ/Wnt pathway activated, and the expression of fibrosis-related factors increased. The above results indicate that subacute CdCl2 exposure increase Cd content in the pig lungs, which leads to M1/M2 imbalance and down-regulates the content of LXA4/FPR2, further activates the oxidative stress/NF-κB/NLRP3 pathway, thereby activating the TGFβ and PPARγ/Wnt pathways to induce fibrosis. This study aims to reveal the toxic effects of CdCl2 and will provide new insights into the toxicology of Cd.

Characteristics of Tibetan pig lung tissue in response to a hypoxic environment on the Qinghai-Tibet Plateau.

To adapt to the plateau environment, Tibetan pigs' lungs have developed a unique physiological mechanism during evolution. The vascular corrosion casting technique and scanning electron microscopy were used to understand arterial architecture. Blood physiological index and quantitative real-time PCR (qRT-PCR) were used for assessing whether the lung can regulate the body through anatomical, physiological and molecular mechanisms to adapt to hypoxic environments. Our study showed that the lungs of Tibetan pigs were heavier and wider and that the pulmonary arteries were thicker and branched and had a denser vascular network than those of Landrace pigs. The hemoglobin (HGB), mean corpuscular hemoglobin concentration (MCHC) values of high-altitude pigs were significantly higher than those of low-altitude pigs. The expression levels of HIF- 1 , EPAS1, EPO and VEGF, but not those of eNOSand EGLN1, were significantly higher in the lungs of high-altitude pigs than in those from pigs at a lower altitude ( ). These findings and a comprehensive analysis help elucidate the pulmonary mechanism of hypoxic adaptation in pigs.

KLF4, a Key Regulator of a Transitive Triplet, Acts on the TGF-β Signaling Pathway and Contributes to High-Altitude Adaptation of Tibetan Pigs.

Tibetan pigs are native mammalian species on the Tibetan Plateau that have evolved distinct physiological traits that allow them to tolerate high-altitude hypoxic environments. However, the genetic mechanism underlying this adaptation remains elusive. Here, based on multitissue transcriptional data from high-altitude Tibetan pigs and low-altitude Rongchang pigs, we performed a weighted correlation network analysis (WGCNA) and identified key modules related to these tissues. Complex network analysis and bioinformatics analysis were integrated to identify key genes and three-node network motifs. We found that among the six tissues (muscle, liver, heart, spleen, kidneys, and lungs), lung tissue may be the key organs for Tibetan pigs to adapt to hypoxic environment. In the lung tissue of Tibetan pigs, we identified KLF4, BCL6B, EGR1, EPAS1, SMAD6, SMAD7, KDR, ATOH8, and CCN1 genes as potential regulators of hypoxia adaption. We found that KLF4 and EGR1 genes might simultaneously regulate the BCL6B gene, forming a KLF4–EGR1–BCL6B complex. This complex, dominated by KLF4, may enhance the hypoxia tolerance of Tibetan pigs by mediating the TGF-β signaling pathway. The complex may also affect the PI3K-Akt signaling pathway, which plays an important role in angiogenesis caused by hypoxia. Therefore, we postulate that the KLF4–EGR1–BCL6B complex may be beneficial for Tibetan pigs to survive better in the hypoxia environments. Although further molecular experiments and independent large-scale studies are needed to verify our findings, these findings may provide new details of the regulatory architecture of hypoxia-adaptive genes and are valuable for understanding the genetic mechanism of hypoxic adaptation in mammals.