Study retrieval and selection
The literature searches and selection procedures are illustrated in Fig. S1. A total of 1,253 studies were collected from different electronic databases. We excluded 523 studies for repeated selection of the same literature. We shortlisted 159 studies related to our study. Finally, 28 studies were selected based on their suitability with the hypothesis of our meta-analysis. The selected literature in our study is characterized in Table S1. The quality of all the selected articles was evaluated using the PEDro scale (Fig. S2). The selected articles were considered of moderate quality for this meta-analysis.
Protein-protein interactions network of HSPs
In a cell, when different entities of a protein bind to each other, the protein becomes functional and plays an important role in the cellular processes (Fig. 1). We used the STRING database to identify and analyze the HSPs as well as to define how HSPs function in different ways 15. HSP90 affects several entities, among which receptor-interacting protein kinase 1 (RIPK1) plays a crucial role in enhancing cell viability by suppressing apoptosis and necroptosis 16. The activator of 90 kDa HSP ATPase homolog 1 (AHSA1) is an important chaperone of HSP90, which regulates osteosarcoma invasion 17. In HSP70, Bcl2-associated athanogene 3 (BAG3) promotes cell adaptability under stressful conditions by stabilizing cytoskeletal integrity 18. In HSP70, Bcl2-associated athanogene 3 (BAG3) promote cell adaptability during stressful condition by making cytoskeletal stability 19. CLPB promotes heat stress resistance activity under heat stress 20. HSP A2 (HSPA2) is a testis-specific protein that increases fertility 21 . TLR4 protein lessens the inflammatory reaction during stress conditions, while HSP60 induces TLR422. SMPD2 reduces stress severity in the endoplasmic reticulum by regulating the cell cycle 23. TRAP-1 regulates oxidative phosphorylation by inhibiting succinate dehydrogenase 24.
Transmembrane region and disorder sequence regulates cycle phase of HSPs expression
The function of a protein depends on its region of origin. This function leads to the translocation of polypeptides into different organelles in the cell. Therefore, transmembrane location is important for protein function. However, we did not find any transmembrane regions in HSP90, HSP70, or HSP60 (Fig. 2A). DNA replication slippage and recombination are responsible for unstable low-complexity regions. However, this does not mean that all the disordered regions contain low-complexity sequences, and secondary structures are also responsible for this region (Fig. 2B). The sequence transformation of HSPs depends on the ligands of the secondary structure of the HSPs. Our results showed that HSP70 and HSP60 possessed higher expression levels than HSP90. HSP90 was expressed in the G1 phase of the cell cycle; HSP70 was expressed during the M and G2 phases; and HSP60 was highly expressed during the G1 phase of the cell cycle (Fig. 2C).
Analysis of studies that investigate HSPs mRNA expression level and study biases
A total of 28 studies reported comparative HSP expressions in different chicken organs under thermoneutral conditions and heat stress. Significant heterogeneity was found in different HSPs in different organs, indicating that the random effect model was I2 = 27%, OR = 0.96, 95% Cl = 0.25, 3.65 of HSP90 in the liver; I2 = 47%, 51%, 64%; OR = 0.58, 0.53, 1.41; 95% Cl = 0.15, 2.26; 0.15, 1.81; 0.41, 4.82 of HSP70 in the brain, heart, and liver, respectively; I2 = 19%; OR = 0.52; 95% Cl = 0.14, 1.93 of HSP60 in the heart. Additionally, the gene expression of HSP70 in the liver was significantly (P < 0.01) different between the thermoneutral and heat stress groups in our study (Fig. 3). We assessed the publication bias of the selected articles using funnel plots. The funnel plot of this study indicates that all the studies were asymmetrical, which means that there was no publication bias except for HSP70 in the liver study publication (Fig. 4).
Relative expression of HSPs mRNA during heat stress
The mRNA expression of HSPs in different organs of chickens was collected from the selected literature. Heat stress changed the mRNA expression compared to the thermoneutral group. We used a coefficient correlation analysis to determine the relationship between the two different organs of the chicken and analyzed the correlation of five different organs with three different HSPs (HSP90, HSP70, and HSP60). For HSP90, muscles with the intestine, as well as heart with the brain, were positively correlated with each other. In contrast, the brain and heart were negatively correlated with muscle, and the intestine was negatively correlated with the brain (Fig. 5A). For HSP70, the liver, intestine, and heart were positively correlated with the brain. In contrast, the liver and intestine were negatively correlated with the heart, while the brain was negatively correlated with the muscle (Fig. 5B). For HSP60, the heart and brain were positively correlated. Nonetheless, the brain and heart were negatively correlated with muscle mass (Fig. 5C).
We performed principal component analysis (PCA) on the expression of HSPs to investigate the relationship between the five different organs of the chicken. For HSP90, the first two principal components (PCs) showed a total variance of 100% among the five organs (PC1= 86.2% and PC2 = 13.8%) (Fig. 5D). The heart, brain, intestine, and muscle were all positively correlated. For HSP70, the first two PCs showed a total variance of 100% among the five organs (PC1 = 57.9% and PC2 = 42.1%). The heart and brain were positively correlated, while the intestine and liver were strongly positively correlated (Fig. 5E). For HSP60, the first two PCs showed a total variance of 100% among the five organs (PC1 = 72.93% and PC2 = 27.07%) (Fig. 5F). The heart and brain were positively correlated.
Categorized the different organs by HSPs mRNA expression
We categorized the different organs of chickens based on the expression of HSP90, HSP70, and HSP60 and their ratios. Circular plots and cluster heat maps were analyzed for different organs. For the HSP90 and HSP70 ratio, the expression ratios were similar in the muscle, intestine, and brain. In contrast, the expression ratio was different in the liver and heart compared to that in the other organs of the chicken (Fig. 6A).
For the HSP90 and HSP60 ratio, the expression ratio was similar to those of HSP90 and HSP70, respectively. For HSP60 and HSP70 ratio, the expression ratio was similar in the muscle and brain. In contrast, the expression ratio was different in the intestine, liver, and heart compared to that in the other organs of the chicken. Furthermore, we categorized the organs according to their relationship to the mRNA expression of HSPs. HSP expression level in the heart was different from that in the other organs (Fig. 6B). The muscle, intestine, and brain tissues were correlated with HSP90, HSP70, and HSP60 mRNA expression.