Morphological examination of broiler jejunum
To evaluate the effect of H2S exposure on autophagy in the jejunum of broilers, the ultrastructural changes of intestinal tissues were observed by transmission electron microscopy. In the control group (Fig. 1A), continuous and complete cell membrane, endoplasmic reticulum, and mitochondrial structure were observed in the intestinal tissue cells. A large number of jejunum cells in the H2S exposure group showed cristae rupture and swelling in the mitochondrion, nuclear atrophy, reduced endoplasmic reticula (indicated by green and blue arrows in Figure 1b), and autophagosomes increased significantly (indicated by red arrow in Figure 1b). These findings clearly showed H2S exposure resulted in autophagy of broiler jejunum cells.
Results of proteomic research, and analysis of biological pathways integrated proteomics and transcriptomics data
To clarify the effect of exogenous H2S exposure on the proteomics of jejunum tissues, we used the TMT isotope method to detect jejunum tissue. Initially, we conducted PCA on the control group data and the H2S group data to show the data's validity. Figure 2A showed the 2D Score Plot in the PCA of proteomics. It can be seen from the figure that the main separation direction of the control group and the H2S group (there are three replicates in each group). The separation between the two groups was obvious, indicating a significant difference in the data expression. Afterward, we can further screen and analyze the data. 8746 proteins were screened according to FDR < 0.01. After a homology search of the identified protein sequences, quantitative proteome analysis was performed. The number of protein groups identified with this project was 3053, among which 2874 satisfied screening criteria. DEPs were identified according to a threshold P-value < 0.05 and |log2 FC| > 0.585. Fig. 2B showed a heat map of 86 DEPs, 55 DEPs of which were up-regulated and 31 DEPs were down-regulated between the control group and the H2S group. To classify DEPS functionally, all DEPS of the H2S group and control group were labeled with GO terms. GO terms divide the function of DEPs into three ontologies: molecular function (MF), cellular component (CC), gene and biological process (BP). Using the GO database can identify the DEPs for the enrichment of function analysis (AdditionalTable 1). Figure 2C showed the GO heat map related to autophagy on proteomics. For cellular components, the major areas of classification were peroxisomal matrix, cotranslational protein targeting to membrane, and establishment of protein localization to endoplasmic reticulum. For biological processes, the major areas of classification were protein targeting to ER, alternative mRNA splicing via spliceosome and collagen metabolic process. For molecular functions, the major areas of classification were collagen binding and protein complex binding. These results suggested that the occurrence of changes in protein metabolism and transport after H2S exposure. Additionally, we used the KEGG pathway database for significance enrichment analysis to obtain the most important biochemical metabolic pathways and signal transduction pathways in which DEPs may be involved. The significantly enriched pathways identified in the KEGG pathway enrichment analysis are shown in Fig. 2D, and pathways related to autophagy such as protein export of pathway have changed significantly.
In order to better analyze the proteomics results, we performed an integrating analysis of proteomics and transcriptomics. The Venn diagram of Figure 2E represented a total of 9 factors in proteomics and transcriptomics with significant differential expression. Most of these factors were involved in the occurrence of ER stress and autophagy. Among them, PKC-δ was significantly expressed and closely related to the expression of other differential factors. Next, GO functional annotation and KEGG signal pathway enrichment analysis were performed on the selected differential factors. The relationship between GO terms was shown in Figure 2F, indicating that protein processing in ER was related to ER stress. In addition, Figure 2G showed that the network diagram of KEGG significantly enriched pathways such as MAPK signaling pathway, FOXO signaling pathway, mTOR signaling pathway and Autophagy, and they were closely related. Based on the above results, a gene interaction network diagram was used to reveal the interaction between differentially regulated factors in the pathway (Figure 2H). Among them, green globules represent the up-regulated genes, and light red globules represent the downregulated genes. The combined analysis showed that the critical factors related to H2S exposure were related to autophagy and ER stress. The results of protein interaction analysis using STRING for selected proteins are shown in Figure 2I. The selected factors related to autophagy and ER stress are also closely related to the protein level.
Detection of miR-181a and PKC-δ in the jejunum tissues of broilers
The biological prediction website TargetScan (http://www.targetscan.org/vert/) predicted that miR-181a and PKC-δ have a targeted relationship in different animals, as shown in Fig. 3A. Therefore, to assess the relationship between miR-181a and PKC-δ after H2S exposure, qRT-PCR was used to detect their expression in the jejunum of broilers. The expression of miR-181a (Fig. 3B) exhibited a trend opposite of PKC-δ (Fig. 3C). Additionally, the level of protein expression of PKC-δ, shown in Fig. 3D, was consistent with the mRNA expression level determined by qRT-PCR. These results suggested that the targeting relationship between miR-181a and PKC-δ was regulated by H2S.
mRNA and protein expression of ER stress related indicators in jejunum tissues of broilers
It is necessary to estimate ER stress in jejunum cells of broilers exposed to H2S. We further studied the mRNA expression levels of ER stress-related genes, XBP1 and GRP78 in broilers exposed to H2S increased significantly (P < 0.05), as shown in Fig. 4A. However, the protein expression levels of XBP1 and GRP78 (Fig. 4B) were consistent with the results of qRT-PCR. The results showed that ER stress had occurred in the jejunum tissues of broilers exposed to H2S.
mRNA and protein expression of JNK, p38 and FOXO1 in jejunum tissues of broilers
To verify the effect of upstream pathway factors on autophagy after H2S exposure, we analyzed the mRNA and protein expression of genes related to the activation of the autophagy pathway (JNK, p38 and FoxO1), as shown in Figure 5. The mRNA and protein levels of JNK, p38 and FOXO1 were significantly up-regulated in the jejunum of broilers under the condition of H2S exposure (P < 0.05). Among the proteins, FOXO1 showed the highest increase in protein expression in the H2S group relative to the control group.
mRNA and protein expression of autophagy-related indicators in jejunum tissues of broilers
To further evaluate the effect of H2S exposure on the autophagy-related signaling pathways in the jejunum of broilers, we detected autophagy-related indicators. The results of qRT-PCR showed that the mRNA levels of Beclin1, LC3, BNIP3, ULK1 and Dynein were significantly increased (P < 0.05), however, those of p62 and mTOR were decreased (P < 0.05) in the H2S group relative to the control group (Fig. 6A). The expression level of LC3II/I, a critical marker of autophagy, significantly increased in the H2S group relative to the control group (P < 0.05). Western blot analysis was used to determine the expression of Beclin1, Dynein, mTOR, BNIP3, ULK1, LC3, and p62 in the jejunum of broilers. Under H2S exposure, the protein levels of autophagy-related indicators were increased, whereas those of p62 and mTOR were decreased (P < 0.05) in Fig. 6B. These results indicated that autophagy occurred in the jejunum of broilers exposed to H2S.