Transcriptome-based biomarker gene screening and evaluation of the extracellular fatty acid-binding protein (Ex-FABP) on immune and angiogenesis-related genes in chicken erythrocytes of tibial dyschondroplasia

Abstract

Background: Tibial dyschondroplasia (TD) is a bone disorder in which dead chondrocytes accumulate as a result of apoptosis and non-vascularization in the tibial bone of broiler chickens. The pathogenicity of TD is under extensive research but is yet not fully understood. Several studies have linked it to apoptosis and non-vascularization in the tibial growth plate (GP). We conceived the idea to find the differentially expressed genes (DEGs) in chicken erythrocytes which vary in expression over time using a likelihood-ratio test (LRT). Thiram was used to induce TD in chickens, and then injected Ex-FABP protein at 0, 20, and 50 μg^.kg^-1 to evaluate its therapeutic effect on 30 screened immunity and angiogenesis-related genes using quantitative PCR (qPCR). The histopathology was also performed in TD chickens to explore the shape, circularity, arrangements of chondrocytes and blood vessels.

Results: Clinical lameness was observed in TD chickens, which decreased with the injection of Ex-FABP. Histopathological findings support Ex-FABP as a therapeutic agent for the morphology and vascularization of affected chondrocytes in TD chickens. qPCR results of 10 immunity (TLR2, TLR3, TLR4, TLR5, TLR7, TLR15, IL-7, MyD88, MHCII, and TRAF6) and 20 angiogenesis-related genes (ITGAV, ITGA2, ITGB2, ITGB3, ITGA5, IL1R1, TBXA2R, RPL17, F13A1, CLU, RAC2, RAP1B, GIT1, FYN, IQGAP2, PTCH1, NCOR2, VAV-like, PTPN11, MAML3) regulated when Ex-FABP is injected to TD chickens.

Conclusion: Immunity and angiogenesis-related genes can be responsible for apoptosis of chondrocytes and vascularization in tibial GP. Injection of Ex-FABP protein to thiram induced TD chickens decrease the chondrocytes damage and improves vascularization.

Keywords: Angiogenesis; Biomarker-genes; Bone-deformity; Broiler; Ex-FABP; Immunity.

Fig. 1

a Analysis of DEGs that show significance in the interaction of time and treatment, using Venn diagrams. There were 249, 393, and 36 DEGs identified on the 6th day vs 2nd day (6T vs 2T), 15th day vs 2nd day (15T vs 2T), 15th day vs 6th day (15T vs 6T). In total 19 DEGs commonly expressed in 6T vs 2T, 15T vs 2T, 15T vs 6T. b Graphical presentation of 19 common DEGs showing the mean of gene expression without time effect on 6th day vs 2nd day (6T vs 2T), 15th day vs 2nd day (15T vs 2T), 15th day vs 6th day (15T vs 6T). Red color represents the control group, and the blue color represents the thiram group

Fig. 2

Gene ontology annotation of differentially expressed genes. a Up-regulated and down-regulated DEGs on 6th day vs 2nd day. b Up-regulated and down-regulated DEGs on 15th day vs 2nd day. c Up-regulated and down-regulated DEGs on 15th day vs 6th day. BP = biological process, CC = cellular process, MF = molecular functions. Counts = number of annoted genes

Fig. 3

KEGG annotation of differentially expressed genes. a KEGG annotation of DEGs up-regulated and down-regulated DEGs on 6th day vs 2nd day. b KEGG annotation of DEGs up-regulated and down-regulated DEGs on 15th day vs 2nd day. c KEGG annotation of DEGs up-regulated and down-regulated DEGs on 15th day vs 6th day

Fig. 4

a Estimation of the equilibrium condition and the tibia parameters in the thiram induced and Ex-FABP supplementing broiler chicken on days 6 and 15. Ex-FABP was injected in group A, B, and C (0, 20, 50 μg^·kg⁻¹ respectively), D, E, and F (0, 20, 50 μg^·kg⁻¹ respectively). Whereas groups D, E, and F gave a diet containing 100 mg^.kg⁻¹ thiram. ^a–f indicates significant differences (P < 0.05). (A) Estimation of the equilibrium condition, (B) tibial dyschondroplasia occurrence score, (C) measurement of growth plate width, (D) measurement of growth plate midline-diameter, (E) measurement of the tibia to toe length, (F) measurement of tibia length. b The clinical and pathological changes in the thiram induced and Ex-FABP supplementing broiler chicken groups of days 6 and 15. Ex-FABP was injected in group A, B, and C (0, 20, 50 μg^·kg⁻¹ respectively), D, E, and F (0, 20, 50 μg^·kg⁻¹ respectively). Whereas groups D, E, and F were given a diet containing 100 mg^.kg⁻¹ thiram. BV, blood vessels; GP, growth plate; TDL, tibial dyschondroplasia lesions; AC, articular cartilage

Fig. 5

a Growth plate histomorphological changes of chondrocytes and blood vessels in the tibia (longitudinal section) for the thiram induced and Ex-FABP supplementing broiler chicken groups of days 6 and 15. Ex-FABP was injected in groups A, B, and C (0, 20, 50 μg^·kg⁻¹ respectively). D, E, and F (0, 20, 50 μg^·kg⁻¹ respectively). Whereas groups D, E, and F were given a diet containing 100 mg^.kg⁻¹ thiram. (A) H&E staining photographs of chondrocytes (Pre-hypertrophic zone) for days 6 and 15. NC, normal chondrocytes; DC, destroyed chondrocytes; RC, recovered chondrocytes. (B) H&E staining photographs of blood vessels (Hypertrophic zone) for days 6 and 15. NV, normal blood vessels; DV, destroyed blood vessels; RV, recovered blood vessels. (C) Zone-wise distribution of growth plate. AC, articular cartilage; PZ, proliferative zone; HZ, hypertrophic zone; CZ, calcification zone. b The histomorphological based quantification of chondrocytes and blood vessels for the thiram induced and Ex-FABP supplementing broiler chicken groups of days 6 and 15 using ImageJ software. Ex-FABP was injected in groups A, B, and C (0, 20, 50 μg^·kg⁻¹ respectively). D, E, and F (0, 20, 50 μg^·kg⁻¹ respectively). Whereas groups D, E, and F gave a diet containing 100 mg^.kg⁻¹ thiram. ^a–f indicates significant differences (P < 0.05). (A) Measurement of chondrocytes area (area=μm). (B) Measurement of chondrocytes area in percentage. (C) Measurement of chondrocytes circularity (area=μm). (D) The relative area of blood vessels. (E) The density of blood vessels

Fig. 6

Expression pattern of angiogenesis and immune-related 30 genes using gel electrophoresis

Fig. 7

a Expression changes of chicken angiogenesis and immune-related 30 genes in the thiram induced and Ex-FABP supplementing broiler chicken groups of day 6. Ex-FABP was injected in groups A, B, and C (0, 20, 50 μg^·kg⁻¹ respectively). D, E, and F (0, 20, 50 μg^·kg⁻¹ respectively). Whereas groups D, E, and F were given a diet containing 100 mg^.kg⁻¹ thiram. ^a–f indicates significant differences (P < 0.05). b Expression changes of chicken angiogenesis and immune-related 30 genes in the thiram induced and Ex-FABP supplementing broiler chicken groups of day 15. Ex-FABP was injected in groups A, B, and C (0, 20, 50 μg^·kg⁻¹ respectively). D, E, and F (0, 20, 50 μg^·kg⁻¹ respectively). Whereas groups D, E, and F were given a diet containing 100 mg^.kg⁻¹ thiram. ^a–f indicates significant differences (P < 0.05)