
==== Front
Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00748-X
10.1016/j.psj.2024.104169
104169
ANIMAL WELL-BEING AND BEHAVIOR
Plasma exosome-derived miR-455-5p targets RPS6KB1 to regulate cartilage homeostasis in valgus-varus deformity (Gallus gallus)
Li Jianzeng *†1
Liu Xinxin *†1
Cai Chunxia *†
Zhang Lujie *†
An Zhiyuan *†
Guo Yujie †
Zhang Yanhua †
Li Wenting *†
Sun Guirong *†
Li Guoxi *†
Kang Xiangtao *†
Han Ruili rlhan@126.com
*†2
⁎ The Shennong Laboratory, Henan Agricultural University, Zhengzhou, 450002, China
† College of Animal Science and Technology, Henan Agricultural University, Zhengzhou 450046, China
2 Corresponding author: rlhan@126.com
1 The authors contributed equally to this work.

11 8 2024
11 2024
11 8 2024
103 11 10416920 5 2024
31 7 2024
© 2024 Published by Elsevier Inc. on behalf of Poultry Science Association Inc.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Valgus-varus deformity (VVD) is a common long bone deformity in broilers. Imbalance in cartilage homeostasis is the main feature of leg disease. Exosomes act as an important intercellular communication vector that regulates chondrogenesis by encapsulating specific nucleic acids and proteins. However, the exact mechanism of how plasma exosomal miRNAs regulate cartilage homeostasis in VVD broilers remains unclear. This study first demonstrated the structural disorder, growth retardation, and reduced proliferative capacity of VVD cartilage in vitro and in vivo. Subsequently, VVD and Normal broiler plasma exosomes were collected for miRNA sequencing. Cartilage-specific miR-455-5p was extraordinarily emphasized by performing bioinformatics analysis on differential miRNA target genes and further validated by tissue expression profiling. PKH67 fluorescently labeled plasma exosomes were shown to be taken up by chondrocytes, deliver miR-455-5p, inhibit chondrocyte proliferation, and disrupt their homeostasis, and these effects could be inhibited by the miR-inhibitors. Mechanistically, MiR-455-5p targets Ribosomal Protein S6 Kinase B1 (RPS6KB1) to inhibit RPS6 phosphorylation and reduce the synthesis of key proteins for cartilage proliferation, which in turn inhibits cartilage proliferation and disrupts its homeostasis. In conclusion, the present study identified abnormalities in VVD cartilage tissue and clarified the specific mechanism by which plasma exosome-derived miR-455-5p regulates cartilage homeostasis.

Key words

Valgus-varus deformity
exosomes
cartilage
miR-455-5p
RPS6KB1
==== Body
pmcINTRODUCTION

In 50 years of high-intensity genetic selection, the growth cycle of broilers has been rapidly shortened, and the weight of broilers has increased by 300% (Knowles et al., 2008; Kierończyk et al., 2017). However, this growth method of “tilting” too much weight makes the skeletal system of broilers immature and unable to withstand such a heavy body, and the broiler leg disease is becoming increasingly prominent (Havenstein et al., 1994). It is estimated that around 1.25 billion poultry worldwide suffer from various leg diseases each year (FAO, 2010), and between 14% and 30% of broilers suffer from lameness (Kestin et al., 1992; Sanotra et al., 2003; Kittelsen et al., 2017). In the United States, the economic cost of bone disease is about $0.8 to 1.2 million each year (Sullivan, 1994; Cook, 2000). In Britain, more than 27% of birds suffer from bone movement disorders, with 3.3% of them barely able to walk (Knowles et al., 2008). In addition, bone disease may indirectly lead to reduced profits during chicken processing (Paz et al., 2010). Among them, noninfectious leg diseases mainly include femoral head necrosis (FHN), tibial dyschondroplasia (TD), and valgus-varus deformity (VVD) (Liu et al., 2023A). In a survey of 4 commercial broiler flocks in southwestern Finland and 50 commercial broiler farms in Norway, approximately 19 to 30% of broilers had gait scores ≥3 for leg disease (Granquist et al., 2019). In another survey of 28 broiler flocks in Denmark with a total of 2800 chicks, the mean prevalence of missing walking ability, TD and VVD was 75.0, 57.1, and 37.0%, respectively (Sanotra et al., 2001). In addition, a recent survey revealed that the overall prevalence of VVD was 1.75%, with 47.5% of cases in males and 52.5% in females (Guo et al., 2019).

Valgus-varus deformity (VVD) is a complex multi-causal long bone deformity common in turkeys and broilers, characterized by varus (inward angulation) or valgus (outward angulation) of the tibiotarsus and tarsometatarsus, bilaterally or unilaterally (Duff et al., 1985; Leterrier et al., 1992; Guo et al., 2019). Due to its special posture, it was initially known as “leg twist,” now abbreviated as VVD (Shim et al., 2012). Unsuitable light, lack of exercise, biomechanics, and chondrodystrophy (caused by micronutrient deficiencies and genetic factors) can all lead to VVD in broilers (Couch et al., 1947; Nestor et al., 1978; Stock et al., 1981; Cruickshank and Sim, 1986). Previous studies have shown that the prevalence of VVD is as high as 1 to 2% and it increases with breeding time, which has a serious impact on broiler welfare and increases economic losses (Newbrey et al., 1988; Guo et al., 2019). Clinical manifestations of VVD in broilers include lameness, pain, decreased feed intake, and lethargy (Tang et al., 2020). In addition, decreased immunity, curvature of long bones, increased brittleness, and decreased bone mass are typical pathologic features (Guo et al., 2019; Li et al., 2023).

Homeostatic imbalance of the cartilage growth plate is one of the most important injury processes in leg disease (Fujii et al., 2022). Chondrocytes in the proliferative phase of the cartilage growth plate secrete the proliferative markers Collagen 2 and Aggrecan into the extracellular matrix to enhance growth plate cartilage stability (Horton et al., 1992). Alkaline Phosphatase (ALP) is a chondrogenic differentiation marker and is regulated by the Runt-related transcription factor 2 (Runx2), and ALP and Runx2 play important roles in chondrocyte calcification and osteoblast differentiation (Vimalraj, 2022). Studies have confirmed that TD and FHN broilers have abnormal cartilage tissue development such as less vascular invasion, dysplasia, impaired mineralization, disturbed chondrocyte arrangement and apoptosis, and significantly reduced Collagen 2, ALP, and Runx2 gene expression (Liu et al., 2021A; Mo et al., 2023; Iqbal et al., 2023). Our group preliminarily found that VVD broiler bone trabeculae were sparse, reduced in strength, and contained white nonmineralized areas (Guo et al., 2019). However, the histopathological changes and growth status of cartilage in VVD broiler growth plates are still unclear.

Exosomes are extracellular vesicles with a diameter of 30 to 150 nm secreted by a variety of cells (Pegtel et al., 2019). They were first discovered and named in reticulocytes in 1983 (Trams et al., 1981). Exosomes begin as early endosomes formed by intracellular traps, regulated by the endosomal network, and are selectively secreted into the blood, tissue fluid, urine, and so on (Kalluri et al., 2020). Vascular substance exchange and endothelial infiltration are the primary modalities regulating cartilage nutrient absorption and growth and development, in which plasma exosomes play an essential role. Exosomes primarily encapsulate a variety of substances that are transported to recipient cells to function as intercellular communication and alter cellular activity, including proteins, mRNA, miRNA, circRNA, lncRNA, DNA, and so on (Raposo et al., 2013). Among them, the most thoroughly studied and the earliest discovered are miRNA, a class of small non-coding RNA molecules, that are 21 to 25 nt in length and negatively regulate gene expression mainly by binding to the 3′UTR region of target genes (Valadi et al., 2007; Lu and Rothenberg, 2018). Early studies have shown that plasma exosomal miRNAs play an important role in enhancing cartilage formation and regeneration, inhibiting cartilage degradation and apoptosis (Meng et al., 2018; Lee et al., 2020; Liu et al., 2023b). Therefore, plasma exosomal miRNAs may be involved in regulating the cartilage homeostasis in VVD broilers, which in turn affects long bone development.

This study firstly elucidated the imbalance of cartilage tissue growth homeostasis in VVD broilers through in vivo and in vitro experiments, identified cartilage-specific miR-455-5p by plasma exosomal miRNA sequencing, bioinformatics analysis and tissue expression profiling, and performed a series of experiments to validate that plasma exosome-derived miR-455-5p targets Ribosomal Protein S6 Kinase B1 (RPS6KB1) to regulate chondrocyte proliferation. This study provides a new perspective and lays a new foundation for broiler leg disease research.

MATERIALS AND METHODS

Ethical Approval

These experiments were performed in strict accordance with the Regulations of the Chinese National Research Council (Dale, 1994) and were approved by the Institutional Animal Care and Use Committee of Henan Agricultural University, China (18-0120).

VVD Broiler Gait Score

Live broilers were laid flat on their wings, the whole leg was immobilized, and VVD was classified into 4 categories based on the angle of the tibiotarsometatarsal joints of the broiler limbs by visual inspection and a protractor. Angulation was classified as (0) normal (angle between tibiotarsus and tarsometatarsus <10°); 1) mild (angle between 10 and 25°); 2) intermediate (angle between 25 and 45°); and 3) severe (angle >45°) (Leterrier et al., 1992; Han den Brand et al., 2022).

Experimental Animals

Fifteen VVD (score = 3, unilateral) and 15 Normal (Nor) male broilers at 5 wk old were randomly selected from a farm (n = 20,000) in Zhongmou, Henan Province. Broilers were raised in 3-layer upright mesh cages with metal cage dimensions of 1 m long x 0.8 m wide x 0.35 m high. The rearing density is 30 chickens from 1- to 9-days-old and 12 chickens from 10- to 37-days-old. The temperature of the initial hatching chicken house is 29 to 33℃, and it is reduced by 2 to 3℃ per week to 21℃ at the age of 4 wk, and the temperature is not lower than 18℃ in the late fattening period. The first 2 wk of the relative humidity of the chicken house is controlled at 65 to 70%, and the later period is controlled at 55 to 60%. Lighting was maintained at 60 lux 24L (0–5 d), 8 lux 17L (6–32 d), and 60 lux 24 L (33–37 d). All broilers were fed uniform diets containing 1.0% Ca, 0.45% P, 2,950 kcal/kg ME, and 21.5% CP at age 1 to 18 d; 0.85% Ca, 0.38% P, 3,100 kcal/kg ME, and 20.0% CP at age 19 to 37 d. All broilers were provided with normal immunization schedules and did not develop Mycoplasma infections.

A sufficient amount of blood was collected aseptically from the wing vein in an anticoagulant tube, centrifuged at 3,500 × rpm for 10 min to obtain plasma, and stored at -80°C for exosome extraction. Then, the broilers were euthanized, and heart, liver, spleen, kidney, duodenum, pectoral muscles, leg muscles, bursa of Fabricius, thymus, and cartilage growth plate tissues were collected and immediately stored at -80°C for subsequent experiments.

Isolation and Identification of Exosomes

Differential centrifugation was used for the isolation and purification of Nor and VVD exosomes (VVD-exos/Nor-exos) from plasma samples. Briefly, 300 × g for 30 min; 2,000 × g for 30 min; and 12,000 × g for 45 min were used to remove cellular debris and protein impurities. A total of 0.22 μm membrane filtration was used for sterilization. A total of 120,000 × g for 2 h; and 100,000 × g for 1 h were used to obtain purified extracellular vesicles, suspended in 100 μL PBS. A transmission electron microscope (Hitachi HT-7700) was used to observe the exosomes, and a nanoparticle size meter (NanoFCM N30E) was used to detect the particle size and purity information of exosomes. Finally, a combination of flow cytometry and western blot were used to detect the expression of exosomal surface marker proteins TSG101, CD9, CD63, and CD81 (Kalluri et al., 2020).

Differential miRNA Screening and Bioinformatics Analysis

Plasma exosomal miRNAs from the VVD and Nor groups were sequenced. miRNA library preparation and sequencing were performed using the Novaseq6000 platform (Novogene, China). Q < 0.05, |log2FC| > 2 was used as screening conditions for differential miRNAs (DEmiRNAs) to ensure the accuracy of sequencing results. TargetScan human (https://www.targetscan.org), miRanda and miRDB (https://www.mirdb.org) were used to predict the target genes of DEmiRNAs Gene Ontology Biological Processes (GO-BP, http://www.geneontology.org), Kyoto Encyclopedia of Genes and Genomes (KEGG, http://www.genome.jp/kegg) analyses were performed on the target genes to select the differential GO-BP and KEGG pathways associated with bone development, and the miRNAs corresponding to the target genes in the differential pathways were statistically analyzed. finally, PPI protein interaction analysis was performed on the target genes of the selected miRNAs. Validation of 6 DEmiRNAs in plasma exosomes by qPCR.

Safranine O-Fast Green Stain

VVD and normal broiler tibial and metatarsal joint growth plates were collected into 5% paraformaldehyde, decalcified 2 d later, paraffin embedded, sectioned and treated with xylene permeabilization, fast green and safranine O solution staining, gradient ethanol dehydration, optical resin for sealing.

Cell Cycle

Chondrocytes grown to 80% were collected for cell cycle assays. In brief, wash using PBS, 1 ml of 0.25% trypsin-EDTA was used to digest the cells, centrifuge at 1,500 × rpm for 5 min, and collect the cells to 1.5 mL EP tubes. Add pre-chilled 75% ethanol 500 μL to the cells fix overnight and store at 4°C. Configure PI/RNase A staining solution in advance, stain the cells after PBS washing, protect from light at room temperature for 30 to 60 min, and record the red fluorescence value at the excitation wavelength of 488 nm.

Vector Construction

Two pairs of 300 to 400 bp DNA single strands were designed with RPS6KB1 targeting sites and mutation sites by combining the miRNA database (https://www.mirbase.org) and NCBI database (https://www.ncbi.nlm.nih.gov) to query the targeting sites of miR-455-5p and RPS6KB1. The PSIcheck2 vector was linearized at NotI and XhoI sites and combined with the designed DNA single strand using the homologous recombination method (Vazyme, Nanjing) to construct WT-RPS6KB1-3′UTR and Mut-RPS6KB1-3′UTR recombinant plasmids for subsequent cell experiments.

Dual Luciferase Reporting System

Chondrocytes grown to 50% were co-transfected with the constructed dual fluorescent vector, mimics-miR-455-5p, inhibitors-miR-455-5p, and its negative control (NC) (Ribobio, Guangzhou) according to the Lip3000 manufacturer's grouping instructions (Thermo, China). After 36 h, luciferase activity was detected separately by a microplate reader and normalized by sea cucumber value as described by the Dual Lucy Assay Kit (Yeasen, Shanghai).

Isolation, Culture, and Transfection of Chondrocytes

Chondrocytes were isolated by 2-step enzyme digestion. In short, the surgical blade separates the cartilage tissue into thin slices, which are cut into 1 mm2 fragments. After pancreatic enzyme digestion for 30 min, digestion was performed overnight with type II collagenase and hyaluronidase, and 100 and 200 mesh cell screens were used to filter impurities. Chondrocytes were cultured in a 5% CO2 incubator at 37°C using a high-sugar DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin solution. The high-sugar medium was replaced every 2 d, and the F2 generation chondrocytes were used for follow-up experiments.

MiR-455-5p-mimics/NC and miR-455-5p-inhibitors/NC were synthesized and transfected into F2 generation chondrocytes with lip3000 reagent. With PBS as the control group, cells were collected 36h later for qPCR to verify transfection efficiency.

Cell Proliferation Assay

CCK-8 and EDU are 2 important indicators for detecting cell viability. The 96-well plate is used for CCK-8. F1 generation chondrocytes were cultured into 96-well plates at a density of 1 × 104 per well, set up at 12 h intervals, set up 7 groups with 6 replicates per group, subsequently, add 10 μL of CCK-8 reagent to each well and incubate for 1.5 h, using a microplate reader (Agilent Synergy LX, Santa Clara, CA) to detect the absorbance at 450 nm. The 24-well plate is used for EDU. In short, chondrocytes grow to 60%, EDU labeling and were fixed in 4% paraformaldehyde, 0.5 TritonX-100 permeabilization, Apollo staining, Hoechst33342 reaction solution staining the nucleus. And finally, observation by fluorescence microscopy (Nikon TS, Tokyo, Japan).

Exosomes Uptake

F1 generation chondrocytes were spread into 24-well plates containing cell creep tablets, and cultured overnight, exosomes free serum was replaced, and plasma exosomes labeled with PKH67 drops were added to chondrocyte supernatant, which was continued to be cultured in 37°C incubator for 3 h, the cell supernatant was discarded, the cells were cleaned with PBS 2 to 3 times, and 4% paraformaldehyde was fixed. The cytoskeleton was labeled with TRITC Phalloidin, and the cell nucleus was stained with an antifade mounting medium with DAPI. The uptake of exosomes was observed under a fluorescence microscope.

qPCR

The total RNA of each tissue was digested and extracted by the Trial method. Nanodrop2000 is used to quantify and normalize total RNA and cDNA. Subsequently, a reverse transcription kit was used to reverse transcribe 1μlRNA into cDNA. qPCR was performed in a 10 μL system, reaction conditions were set to 40 cycles of 95°C for 30 s, 95°C for 10 s, 60°C for 30 s; 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. All primers are synthesized by SunYa and Tsingke and are detailed in Table S1.

Western Blot

The total protein of chondrocytes was extracted by RIPA Lysis Buffer (Epizyme, Shanghai, China) mixture with protease inhibitor and phosphatase inhibitor, and the protein was quantified by BCA kit (Epizyme, Shanghai, China). The proteins were isolated by 10% SDS-PAGE gel electrophoresis and transferred to 0.22μm PVDF membrane. TBST of 5% skim milk powder was isolated for 2 hours, and then incubated in rabbit polyclonal antibodies TSG101, CD81, CD9, COL2A1, RPS6, RPS6KB1, and P-RPS6 overnight. TBST was cleaned 3 times, and the PVDF membrane was incubated with the second antibody of goat and rabbit at room temperature for 1h. Color development was performed using an ultra-sensitive chemiluminescence kit PVDF membrane and images were captured using an imaging system.

Statistical Analysis

All data were statistically analyzed using a t-test (2 groups) and one-way ANOVA (multiple groups) in SPSS 24 (SPSS for Windows, Standard version 24.0; SPSS). The results of the analysis were presented using GraphPad Prism 9.0. P < 0.05 (*), p < 0.01 (**) was considered statistically significant.

RESULTS

Abnormal Cartilage Development in VVD Broilers

Chondrocyte disorganization due to chondrodystrophy is one of the main pathological phenotypes of VVD in broilers (Wolbach et al.,1953, Wise et al.,1973, Whitehead, 1997). To investigate the growth status of cartilage in VVD broilers, The tibial and metatarsal growth plates were stained in Normal (Nor) and VVD groups with Safranine O-Fast Green. The results indicate that, relative to the Nor group, the growth plate in the VVD group had fewer blood vessels, thinner proliferation zones, fewer super-recruited osteoblasts and delayed calcification. In the Nor group, the hypertrophic chondrocytes were densely and compactly distributed with a neat structure, while in the VVD group, the hypertrophic chondrocytes were disorganized, and most of the chondrocytes showed vacuole-like nuclear consolidation and nucleolus lysis (Figure 1A). In addition, qPCR results showed that the cartilage Collagen 2, ALP and Runx2 mRNA expression levels were significantly lower in the VVD group relative to the Nor group (P < 0.01) (Figure 1D).Figure 1 Abnormal chondrogenesis in VVD broilers by in vitro and in vivo experiments. (A) Saf-ranine O-Fast Green Staining of articular cartilage growth plates of the proximal tibia and proximal metatarsal; AC, articular cartilage; BV, blood vessels; PZ, proliferative zone; HZ, hyper-trophic zone; DZ, Calcified zone; (B) One-week growth record of primary chondrocytes (10x); (C) Gene expression of primary and F1 chondrocytes (*P < 0.05, ** P < 0.01); (D) Gene expression of cartilage tissue in 5 wk (*P < 0.05, ** P < 0.01); (E) Cell cycle of VVD and normal broilers (*P < 0.05, ** P < 0.01).

Figure 1

In vitro isolation and culture of Nor and VVD chondrocytes revealed that VVD chondrocytes had slow growth, weak apposition ability, and increased number of cell death (Figure 1B). The chondrocytes of the Nor group grew to 90% as a node to explore the proliferation ability of chondrocytes in both groups. QPCR results indicated that the mRNA expression level of Collagen 2 in F0 and F1 generation chondrocytes of VVD group was significantly higher than that of the Nor group (P < 0.01) (Figure 1C). The cell cycle results showed that the number of S-phase cells in the VVD group was significantly higher than that in the Nor group (P < 0.01), that is, chondrocytes in the VVD group grew slower than chondrocytes in the Nor group (Figure 1E). These combined in vivo and in vitro, results suggest that VVD cartilage is structurally disturbed, growth retarded, and has diminished proliferative and differentiation capacity that is, chondrodysplasia.

Characteristics of Plasma Exosomes in the VVD and Nor Groups

Plasma exosomes from the VVD and Nor groups were purified by classical differential centrifugation. Transmission electron microscopy indicates that the plasma exosomes in both the VVD and Nor groups have a typical concave disc-like structure (Figure 2A). NTA analysis showed that in plasma exosomes from the Nor group, vesicles with a particle size of 30 to 150 nm accounted for 99.20% of the total particles and a concentration of 9.44 × 109/mL, whereas in exosomes from the VVD group, vesicles with a particle size of 30 to 150 nm accounted for 99.34% of the total particles and a concentration of 7.63 × 109/mL (Figures 2B and 2C). Flow cytometry showed positive expression of exosomal surface proteins CD9, CD63 and CD81 at 13.4, 12.3, and 13.1%, respectively (Figure 2E), and western blot showed positive for TSG101, CD9, and weakly positive for CD81 (Figure 2D). Together, these results showed no significant differences between the 2 groups and were consistent with previous identification reports, confirming the present successful isolation of exosomes from plasma.Figure 2 Identification of plasma exosomes in VVD and Nor broilers. (A) Transmission electron microscopy in VVD and Nor plasma exosomes; (B, C) NTA particle size analysis in VVD and Nor plasma exosomes; (D) Western blot detected exosome-labeled proteins TSG101, CD9, and CD81; (E) Flow cytometry detected exosome-labeled proteins CD9, CD63, and CD81.

Figure 2

Plasma Exosomes Inhibit Chondrocyte Proliferation and Disrupt Homeostasis

Fluorescence microscopy imaging showed that chondrocytes contained green plasma exosomes, confirming that plasma exosomes could be taken up by chondrocytes (Figure 3A). At the same time, to explore whether plasma exosomes affect cartilage growth and development, chondrocytes were isolated and cultured and assessed for plasma exosome interference concentrations. The results showed that the optimal interference concentration of plasma exosomes was 6.94 μg/mL (Figure 3B). Chondrocytes were treated with PBS, Nor-exos, VVD-exos, and Nor/VVD-exos-inhibitors/NC at the optimal concentration and time, and the results showed that the gene expression levels of Collagen 2 and MMP 9 were significantly decreased (P < 0.01) in Nor-exos and VVD-exos groups, while the gene expression levels of MMP13 were increased, with a significant level in the VVD-exos group (P < 0.01) (Figure 3C). These results suggest that plasma exosomes can be taken up by chondrocytes and inhibit chondrocyte proliferation and disrupt cartilage homeostasis.Figure 3 Plasma exosomes transport miR-455-5p to inhibit chondrocyte proliferation. (A) PKH67-labeled exosomes are taken up by chondrocytes; (B) Optimal concentration for exosome transfer into chondrocytes; (C) Chondrocyte proliferation and homeostatic gene expression in different subgroups (*P < 0.05, ** P < 0.01).

Figure 3

Plasma Exosomal miRNA Sequencing and Bioinformatics Analysis

Three mixed samples each of Nor and VVD plasma exosomes were collected for miRNA-seq. A total of 18 differential miRNAs (DEmiRNAs) were screened, including 9 up-regulated and 9 down-regulated, and the DEmiRNAs were well clustered (Figures 4C and 4D). TargetScan human, miRanda and miRDB predicted 1615 candidate target genes for these 18 DEmiRNAs. The GO-BP enrichment showed that the target genes were mainly anatomical structure morphogenesis, movement of cell or subcellular component, cell morphogenesis, cell projection organization and protein dephosphorylation. KEGG was concentrated in 137 pathways, mainly including protein synthesis and degradation, autophagy and cell growth and development, such as MAPK signaling pathway, hedgehog signaling pathway, insulin signaling pathway, TGF-β signaling pathway and mTOR signaling pathway (Figures 4B and 4F). Six miRNAs were randomly selected for qPCR validation, the results found that, in particular, the expression level of gga-miR-455-5p in VVD-exos is significantly lower than that in Nor-exos (P < 0.05). The qPCR validation results of these 6 miRNAs are consistent with the sequencing results. Demonstrating the accuracy of the sequencing data in this study (Figure 4H).Figure 4 Plasma exosomal miRNA sequencing, bioinformatics analysis and screening of key miR-455-5p in VVD and Nor broilers. (A) Differential miRNA GO-BP enrichment weight; (B) The top 20 most differential GO-BP terms; (C) Plasma exosomal DEmiRNA volcano maps in VVD and Nor broilers; (D) Plasma exosomal DEmiRNA cluster plots in VVD and Nor broilers; (F) The top 10 most differential KEGG signaling pathways; (G) PPI protein interaction maps of miR-2188-5p, miR-455-5p and miR-7b; (H) qPCR validation of 6 differential miRNAs (*P < 0.05, ** P < 0.01).

Figure 4

Plasma Exosomes Contained Critical miR-455-5p and Transferred miR-455-5p to Chondrocytes

Bone-associated GO-BP and KEGG pathway-associated genes were selected for weighted analysis of DEmiRNAs (Figures 4A, 4E), and were plotted gene-DEmiRNAs interactions, and the synthesis indicated that gga-miR-455-5p had a high weight ratio in VVD chondrodysplasia and may play an important role in chondrodysplasia (Figure 4G). In particular, miR-455-3p and miR-140 were confirmed to be specifically expressed in long bones during embryonic development, and gradually focused on joints and cartilage as the embryo developed (Swingler et al., 2012). In this study, multi-tissue expression profiling showed that miR-455-5p was extremely significantly higher in cartilage tissues than in other tissues (P < 0.01) (Figure 5A), and the specificity of miR-455-5p expression in broiler cartilage was preliminarily demonstrated. Therefore, miR-455-5p was used as a critical miRNA for subsequent experiments. Treatment of chondrocytes with VVD-exos and Nor-exos revealed that both of them up-regulated miR-455-5p, with Nor-exos having a stronger up-regulatory effect. This trend decreased after miR-inhibitors combination treatment (Figure 5B). In short, plasma exosomes contain critical miR-455-5p and can transfer miR-455-5p into chondrocytes.Figure 5 MiR-455-5p regulates cartilage homeostasis. (A) Tissue expression profile of miR-455-5p (*P < 0.05, ** P < 0.01); (B) miR-455-5p expression in different subgroups of chondrocytes (*P < 0.05, ** P < 0.01); (C) qPCR detection of chondrocyte miR-455-5p expression after miR-455-5p-mimics/inhibitors treatment (*P < 0.05, ** P < 0.01); (D) qPCR detection of chondrocyte proliferation-related gene expression after miR-455-5p-mimics/inhibitors treatment (*P < 0.05, ** P < 0.01); (E) Western blot detection of chondrocyte Collagen 2 protein expression after miR-455-5p-mimics/inhibitors treatment (*P < 0.05, ** P < 0.01); (F, G, H, M) EDU detection of chondrocyte proliferation after miR-455-5p-mimics/inhibitors treatment (* P < 0.05, ** P < 0.01); (I, L) CCK-8 detection of chondrocyte proliferation after miR-455-5p-mimics/inhibitors treatment; (J, K) qPCR detection of chondrocyte cycle-related gene expression after miR-455-5p-mimics/inhibitors treatment (*P < 0.05, ** P < 0.01).

Figure 5

MiR-455-5p Inhibits Chondrocyte Proliferation and Disrupts Homeostasis

Next, to reveal the relationship between miR-455-5p and VVD cartilage homeostatic imbalance. The qPCR results indicated that miR-455-5p levels were significantly up-regulated in the miR-455-5p mimic-treated group (P < 0.01), and miR-455-5p-inhibitors-treated group significantly inhibited endogenous cellular miR-455-5p levels (P < 0.01) (Figure 5C). The expression levels of the chondrogenic proliferation and homeostasis marker genes Collagen 2, MMP9 and MMP13 in miR-455-5p-mimics-treated group were significantly down-regulated (P < 0.01). The expression levels of Collagen 2 and MMP9 genes in the miR-455-5p-inhibitors-treated group were significantly up-regulated (P < 0.01) (Figure 5D). Western blot focused on exploring the expression of Collagen 2 protein and showed that its expression was decreased in miR-455-5p-mimics-treated group and increased in miR-455-5p-inhibitors-treated group (Figure 5E). Combined with EDU, CCK-8, and cell cycle qPCR results (Figures 5F–5M), the combined results showed that miR-455-5p-mimics inhibited chondrocyte proliferation, and conversely, miR-455-5p-inhibitors partially promoted chondrocyte proliferation.

MiR-455-5p and RPS6KB1 Targeting Validation

Considering that miRNAs often act on their target genes, Targetscan Human, miRanda, and miRDB were used to predict the possible target genes of miR-455-5p alone and performed KEGG analysis to try to identify key genes that affect bone growth and development pathway. After careful review of these studies, the RPS6KB1 gene with Targetscan Human7.2 score 86, miRanda score 147, and MFE: -24 kal/mol was highlighted (Figure 6A). Predicted targets were further revealed that only gga-miR-455-5p had a unique target with gga-RPS6KB1, compared to other species (Supplementary word S1). KEGG analysis showed that RPS6KB1 was involved in autophagy, mTOR, RrbB, TGF-β and insulin signaling pathways related to bone growth and development (Figure 6C). The RPS6KB1 wild and mutant vectors were further designed to explore whether it is a key target gene mediating the dysregulation of cartilage homeostasis in VVD. In chondrocytes and DF1 cells, miR-455-5p could not bind to the complementary region of the target gene to inhibit luciferase activity when the target region was mutated (Figures 6D and 6E). The qPCR results show that the expression of RPS6KB1 in chondrocytes treated with miR-455-5p-mimics was significantly decreased, while the expression of RPS6KB1 in chondrocytes treated with miR-455-5p-inhibitors was significantly increased (Figure 6B). The above results confirm the targeted regulatory effects of miR-455-5p and RPS6KB1.Figure 6 Validation of miR-455-5p in relation to RPS6KB1 targeting. (A) Intersection of predicted scores for miR-455-5p and RPS6KB1 targeting relationships from Targetscan Human, miRanda, and miRDB websites; (B) qPCR detection of chondrocyte RPS6KB1 gene expression after miR-455-5p-mimics/inhibitors treatment (* P < 0.05, ** P < 0.01); (C) Analysis of miR-455-5p target gene KEGG pathway; (D) miR-455-5p and RPS6KB1 targeting sites; (E) Validation of miR-455-5p and RPS6KB1 targeting relationship in chondrocytes and DF1 cells using a dual luciferase reporter system (*P < 0.05, ** P < 0.01).

Figure 6

MiR-455-5p Targets RPS6KB1 to Inhibit Collagen 2 Synthesis

To investigate whether RPS6KB1 is one of the key target genes regulating chondrocyte proliferation and homeostasis, 3 small interfering RNAs for RPS6KB1 (siRPS6KB1-1/2/3) and their siNC were synthesized in vitro to treat F2 generation chondrocytes. The results of qPCR and western blot showed that siRPS6KB1-1 interfered with 40% successfully, and siRPS6KB1-2/3 had no significant change, so siRPS6KB1-1 was used for follow-up experiments (Figure 7A). The gene expression levels of Collagen 2 and MMP13 in siRPS6KB1 treatment group were significantly increased (P < 0.01) (Figure 7C). The protein expression of Collagen 2, RPS6, P-RPS6, P-RPS6/RPS6 was significantly decreased (P < 0.05) (Figures 7D, 7F–7J). The results of EDU and CCK-8 showed that siRPS6KB1 significantly reduced the cell proliferation rate from 12 to 48 h (P < 0.01) (Figures 7B, 7E, and 7H). It was comprehensively demonstrated that siRPS6KB1 inhibited the proliferation of chondrocytes and disrupted their homeostasis.Figure 7 RPS6KB1 inhibits chondrocyte proliferation. (A) QPCR assay of RPS6KB1 interference efficiency (*P < 0.05, ** P < 0.01); (B, E) EDU detection of chondrocyte proliferation after siRPS6KB1 treatment; (C) qPCR detection of chondrocyte proliferation-related gene expression after miR-455-5p-mimics/inhibitors treatment (*P < 0.05, ** P < 0.01); (D) Western blot detection of chondrocyte Collagen 2 protein expression after siRPS6KB1 treatment; (H) CCK-8 detection of chondrocyte proliferation after siRPS6KB1 treatment; (F, G, J, I) Western blot detection of chon-drocyte RPS6KB1, p-RPS6, RPS6 protein expression after miR-455-5p-mimics/inhibitors or siRPS6KB1 treatment (*P < 0.05, ** P < 0.01).

Figure 7

Subsequently, the western blot results showed that Collagen 2, RPS6, P-RPS6, P-RPS6/RPS6 protein expression were significantly decreased (P < 0.05/0.01) (Figure 7I), which was consistent with the siRPS6KB1 results. This confirmed that miR-455-5p inhibited the phosphorylation of RPS6 by targeting RPS6KB1, which in turn decreased the translation process of the protein.

DISCUSSION

VVD leg disease in broilers is a typical “side effect of long bone deformity” in rapidly growing broilers. It has a high incidence and often develops at the early stage of broilers' growth, causing important economic losses to the breeding industry (Shim et al., 2012). Our research group has previously confirmed that the long bones of VVD broilers are curved, with decreased bone mineral density and weakened bone strength (Kaukonen et al., 2017). Cartilage homeostasis is critical for long bone growth and development. Therefore, this study highlights the pathological changes in the cartilage growth plate of VVD broilers in vivo and in vitro. The results showed that the cartilage tissues of tibia and metatarsus of VVD broilers were structurally disorganized with retarded growth and diminished proliferation, whereas the region of cartilage growth plate of broilers in Nor group was more vascularized with dense distribution of osteoblasts and regular structure. Earlier studies showed that TD broiler chondrogenic growth plate had significantly weaker vascular invasion, dispersed distribution of osteoblasts in the hypertrophic area, and most osteoblasts and chondrocytes showed nuclear consolidation and nucleolysis (Xu et al., 2023). After isolation and culture of TD broiler cartilage growth plates in vitro for 24 d, the protein content of TD chondrocytes was found to be significantly lower and the ALP activity was significantly lower than that of healthy articular chondrocytes (Wu et al., 2005). Up-regulation of BMP6 gene expression alleviated the abnormal growth of TD chondrocytes (Lu et al., 2021). In broiler FHN, healthy cartilage tissue HE-stained chondrocytes had normal morphology and intact structure, while the methylprednisolone-treated group had more tissue gaps, uneven cell distribution, increased vesicles, and greater damage to cartilage structure in the heavily methylprednisolone-treated group (Yu et al., 2020; Liu et al., 2021B), and could significantly increase apoptosis of chondrocytes (Yu et al., 2022A). The results of the present study were similar to the histopathological phenotypes of cartilage in non-infectious leg diseases such as TD and FHN, but their symptoms were relatively mild, probably due to spontaneous generation of leg disease.

Exosomes maintain normal physiological processes primarily through specific uptake by target cells and play an important role in the development of disease (Pegtel et al., 2019; Kalluri et al., 2020). This study demonstrated that plasma exosomes can be taken up by cells through qPCR, CCK-8 toxicity assay and PKH67 fluorescent labeling of exosomes. and can inhibit cell proliferation and disrupt their homeostasis. Exosomes contain proteins, DNA, miRNA, LncRNA, circRNA, metabolites and so on. This study showed that Nor-exos group had a weaker effect on inhibiting the proliferation and disrupting the homeostasis of chondrocytes than VVD-exos group, which may be the result of the joint action of miRNA, protein and even circRNA in exosomes. Among them, miRNA is the earliest discovered and most thoroughly studied class of exosome contents, and most studies have shown that exosome-delivered specific miRNA can significantly affect the function of target cells. Studies have shown that plasma exosomal miR-708-5p from fracture patients targets SSRP1 and inhibits osteogenesis of BMSCs by suppressing Wnt signaling (Yu et al., 2022B). Previous study has found that the plasma exosome miR-204-5p mediates communication between immune cells and synovial fibroblasts and plays an important role in rheumatoid arthritis (Wu et al., 2022). Furthermore, human umbilical cord blood exosome miR-3960 ameliorates bone loss in senile osteoporotic mice by stimulating bone formation and inhibiting bone resorption (Hu et al., 2019). Therefore, plasma exosome-derived miRNAs might be involved in the regulation of cartilage growth and development in VVD and healthy broilers. miRNA is a 15 to 25 nt small molecule RNA, and as an important regulatory element of the organism, it plays a unique role in regulating cell growth and development (Lu and Rothenberg, 2018; Swingler et al., 2019). This study sequenced plasma exosomal miRNAs and identified 18 differential miRNAs after screening. GO-BP was mainly for cell morphological changes and development, KEGG was mainly enriched in protein synthesis, MAPK, PI3K/AKT/mTOR, insulin, TGF-β and other cell growth-related signaling pathways, of which most studies have confirmed that the PI3K/AKT/mTOR pathway is required for cartilage homeostasis. Studies has shown that fluoride, and epimedium decreased chondrocyte proliferation and increased apoptosis and autophagy in rats by inhibiting the PI3K/AKT/mTOR signaling pathway (Ma et al., 2021; Tang et al., 2021). miR-7/EGFR/MEGF9 axis regulates osteoarthritic cartilage degradation via PI3K/AKT/mTOR signaling pathway (Jiang et al., 2021). Concomitantly, through Log2FC, PPI protein interactions and comprehensive miR-455-5p tissue expression profiles, miR-455-5p was focused on. Additionally, previous studies have shown that miR-455 mainly regulates TGF-β and PI3K/AKT/mTOR signaling pathways (Swingler et al., 2012; Wen et al., 2020), this study selected miR-455-5p for in-depth study.

Notably, mature mou-miR-455-5p is 5′-UAUGUGCCUUUUGGACUACAUCG-3′, whereas mature gga-miR-455-5p is 5′-UAUGUGCCCUUUGGACUACAUCG-3′, with a single base change. Therefore, an in-depth study of gga-miR-455-5p on cartilage homeostasis in VVD broilers is essential. The study demonstrated for the first time that miR-455-5p was also specifically highly expressed in poultry cartilage tissues, and the expression in the cartilage tissues of VVD broilers was significantly higher than that of healthy broilers. Notably, the plasma exosomal miR-455-5p expression obtained by sequencing in this study was inconsistent with subsequent cellular experiments. Firstly, Plasma exosomal miRNAs are a complex mixture (Arraud et al., 2014; Yu et al., 2016; Kalluri et al., 2020). VVD is a syndromic leg disease (Cruickshank et al., 1986; Tang et al., 2020). Pathologic changes in other organs are also likely to be reflected in plasma and its exosomal miRNA expression. Secondly, Previous studies have shown that the active sorting mechanism of some exosomal miRNAs is tissue-specific; that the exosomal miRNA profile may be different from the parental cellular miRNA profile, and that the intracellular miRNA pool is the sum of endogenous miRNA production and exogenous circulating miRNAs (Zhang et al., 2015; Mori et al., 2019; Garcia-Martin et al., 2022). In this study, miR-455-5p was clearly transcribed in cartilage by tissue expression profiling. It cannot be excluded that, cartilage acts as a parent tissue releasing exosomes encapsulating miR-455-5p into plasma. Undeniably, the present study is not the same as conventional studies in which the lesion tissue under study is by default the target tissue for one of the miRNAs in plasma exosomes (Li et al., 2021; Sun et al., 2022). However, the present study gives new insights into the origin and targeting of small molecules carried by plasma exosomes in integrative diseases.

To clarify the intrinsic relationship between miR-455-5p and cartilage growth and development, mimics/inhibitors and its NC were designed to treat F2 generation chondrocytes. Overexpression of miR-455-5p significantly inhibited cell proliferation and destroyed its homeostasis, while interference of miR-455-5p had the opposite effect. Early studies on human/mouse cartilage found that miR-455-3p can target PTEN to reduce MMP13 and increase Collagen 2 protein and gene expression through the PI3K/AKT signaling pathway (Wen et al., 2020). LncRNA HOTTIP can adsorb miR-455-3p and promote CCL3 gene expression to induce cartilage degradation (Mao et al., 2019). Both miR-455-5p and -3p inhibit HIF-2α expression and cooperate to regulate cartilage homeostasis (Ito et al., 2021). This study for the first time investigated the function of miR-455-5p in poultry chondrocytes and showed that miR-455-5p mainly inhibited the proliferation of chondrocytes in poultry chondrocytes, which corresponded to the thinning of the proliferative zone of the early cartilage tissue and the slow growth of chondrocytes.

After that, this study demonstrated that miR-455-5p and RPS6KB1 have obvious targeting relationship on chondrocytes and DF1 cells by using the dual luciferase reporter system. The miR-455 family mainly acts on typical cell growth and development pathways, such as PI3K/AKT/mTOR, and TGF-β (Swingler et al., 2012; Wen et al., 2020). RPS6KB1 is ribosomal protein S6 kinase B1, and its upstream is the key protein mTOR, which activates RPS6KB1 through phosphorylated mTOR and then activated RPS6KB1 phosphorylates its downstream molecules RPS6 and EIF4B, thereby controlling the downstream process of protein synthesis during cell growth (Sridharan et al., 2020; Hwang et al., 2021). Therefore, by interfering the expression of RPS6KB1 gene, this study explored the regulation of RPS6KB1 on the phosphorylation level of downstream proteins. The results showed that RPS6KB1 decreased the protein content of Collagen 2, a marker of chondrocyte proliferation, by decreasing the phosphorylation of RPS6 protein. Collagen 2 gene expression level increased. Then, siRPS6KB1 may only inhibited the translation initiation process of cellular proteins, resulting in a decrease in Collagen 2 protein and an increase in gene expression. In addition, to complete the argument, this study also detected the changes in the phosphorylation levels of miR-455-5p-mimics/inhibitors, and the results were consistent with those of RPS6KB1. That is, miR-455-5p inhibits RPS6 protein phosphorylation by targeting RPS6KB1, inhibits chondrocyte proliferation, and disrupts its homeostasis (Figure 8).Figure 8 Mechanism pattern of plasma exosome-delivered miR-455-5p regulates RPS6 phosphor-ylation by targeting RPS6KB1 and thus affects chondroprotein synthesis.

Figure 8

CONCLUSIONS

In summary, this study identified dysplasia of VVD cartilage tissue and found that cartilage-specific miR-455-5p, a key in plasma exosome transport, targets RPS6KB1 to inhibit RPS6 phosphorylation and reduces the synthesis of key proteins for cartilage proliferation, which in turn inhibits cartilage proliferation and disrupts its homeostasis. This study investigated the pathological changes of VVD cartilage for the first time, and used exosomes in the study of broiler leg disease, and explored the influence of exosome miRNA on the development of broiler cartilage, providing a new direction for the study of VVD leg disease.

DISCLOSURES

The authors declare no conflicts of interest.

Appendix Supplementary materials

Image, application 1

ACKNOWLEDGMENTS

This work was supported by the Key Research Project of the Shennong Laboratory (SN01-2022-05 ), Natural Science Foundation of Henan Province (no. 222300420458 ) and Scientific Studio of Zhongyuan Scholars (no. 30601985 ).

Data Availability Statement: The data were submitted to the Genome Expression Omnibus (Accession Numbers PRJNA932579) in NCBI.

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2024.104169.
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