Transcriptome-based protein-protein interaction analysis reveals immune gene network elucidating white body immunity mechanisms in response to LPS stimulation in Amphioctopus fangsiao

Highlights • This study integrates protein-protein interaction network into transcriptome analysis for the first time in cephalopods.• The result lays a function for further exploring invertebrate LPS stress at the molecular level.• It provides valuable gene resources for further study of the white body immune responses in pathogen-infected invertebrate.


Introduction
Invertebrates possess a sophisticated immune system, which has garnered significant scholarly attention [1].As one of the immune organs in cephalopods, white body were discovered as early as 1984 in Northern shortfin squid (Illex illecebrosus) and were considered as hematopoietic organs in cephalopods in subsequent studies, which may be involved in important functions such as immune regulation, hematopoietic activation, and pathogen infection [2].For example, the white body of cephalopods contain blood cells at different stages of development [3].In addition, white body obtain arterial blood from several branches of the ophthalmic artery and form a complex network of capillaries within the organ [4].This network not only contributes to the maturation of blood cells, but also facilitates the entry of mature blood cells into the circulatory system through the capillaries, thereby participating in immune responses and other immune activities.Hemocytes in the white body are produced by leukocytes, which play a key role in a variety of cellular immune processes including phagocytosis, lysis, and attack, thus becoming an important component of the innate immune system [3,5].
The cephalopod Gold-ringed octopus (Amphioctopus fangsiao), popular among consumers for its flavorful meat and high arginine content, is a traditional dietary in coastal areas of China [6,7].There has been a recent surge of interest in the artificial cultivation of gold-ringed octopus, A. fangsiao, but relevant research endeavors have been considerably hindered by disease outbreaks brought on by pathogenic infestations [8,9].Seawater hosts a majority of gram-negative bacteria [10], several species of which have been identified to be harmful to various marine organisms such as Pacific oyster (Crassostrea gigas) [11], Horsehair crab (Portunus trituberculatus) [12], Japanese flounder (Paralichthys olivaceu) s [13], Pacific white shrimp (Penaeus vannamei) [14], and Green-lipped mussel (Perna canaliculus) [15].Lipopolysaccharide (LPS) is a major component of the cell wall of Gram-negative bacteria and consists of O-antigens, core oligosaccharides, and covalently linked lipid A, which is the main active center of LPS [16].In the host's immune system, LPS is recognized by immune cells as pathogen-associated molecular patterns (PAMPs) and initiates the immune response process upon invasion by Gram-negative bacteria [17].This recognition process triggers immune signaling pathways, which in turn stimulate host defense mechanisms.
RNA-seq presents a potent approach towards deciphering gene transcription profiles inherent to specific biological processes [18,19].Recently, this technique has proven valuable in investigating the molecular mechanisms crucial for immunity [20], culminating in the discovery of a myriad of immunity-related genes across diverse organisms such as Jamaican fruit bat (Artibeus jamaicensis) [21], Yesso scallop (Patinopecten yessoensis) [22], and Bearded fireworm (Hermodice carunculata) [23].Previous studies on Gram-negative bacterial infections have typically focused on individual genes [24][25][26].While these single-gene studies enrich scientific knowledge, their scope of research remains inherently restrictive.Notably, genes do not act independently; instead, they frequently participate in numerous regulatory pathways, influencing cellular processes through the formation of expansive functional networks [27].Creating an immune interaction network to identify crucial network nodes provides a strategic method for pinpointing hub genes that are instrumental in immune responses.
In the present study, the transcriptional profiles of A. fangsiao's white body tissue were examined following LPS injection stimulation, with assessments conducted at time points of 6 and 24 hours.Transcriptome sequencing was performed using the Illumina HiSeq 6000 platform, and the transcriptome data were subsequently screened for differentially expressed genes using the DESeq2 method based on negative binomial distribution.Through GO and KEGG functional enrichment analyses, we dug deeper into the important genes involved in the A. fangsiao immunization process.In addition, we constructed protein-protein interaction (PPI) networks to identify hub DEGs associated with LPS infection.Finally, the screened 20 hub genes were verified by qRT-PCR to ensure their consistency with the RNA-Seq results.These findings provide important clues to our in-depth understanding of the immune response of A. fangsiao, and also provide a strong reference for future related studies, thus promoting the study and understanding of the function of the cephalopod immune system.

Sample preparation
We collected A. fangsiao specimens from the Yellow Sea area of Yantai City, Shandong Province, China.To ensure the stability of the samples during the experiment, we placed the specimens in five temporary tanks, each measuring 70.4 cm in length, 52.5 cm in width, and 87.4 cm in height.These tanks are filled with oxygen-rich seawater and are maintained in a temperature range of 18 to 20 • C. The water quality indicators for farmed seawater include: dissolved oxygen = 5.4 mg/L, pH = 8.2, and salinity = 30 ± 0.5 ppt.The qualitative indicators remained stable throughout the experiment; this was also true for the seven days prior to the start of the experiment.To mitigate potential stress, we put food and ceramic jars in the tank.Healthy, vigorous specimens of A. fangsiao with an average age of about five months and an average weight of 34.7 g were purposely selected for subsequent experiments.The experimental samples were divided into three groups, a blank group without any treatment (AB), a control group injected with 100 μLPBS (AP), and an experimental group injected with the same  volume of LPS (AL).The injections were administered at the first left wrist, where it touched the wrist.Group AB used sterilized dissection tools to dissect carcass parts of Fanshawe pigs Group AB used sterilized dissection tools to dissect carcass parts of A. fangsiao, removed white body tissues, and immediately froze them in liquid nitrogen and stored them at -80 • C for subsequent analysis.The same operations of two other groups happened at 6 h and 24 h post-injection, respectively.

The extraction of RNA
In each of the above three treatment groups, nine gold-ringed octopuses were randomly selected for RNA extraction: 0 h sampling (C0h) for group AB, 6 h sampling (P6h) for group AP, 24 h sampling (P24h) for group AP, 6 h sampling (L6h) for group AL, and 24 h sampling (L24h) for group AL.From the nine A. fangsiao specimens, equal molar masses of three random samples were combined to create a single replicate [28].This process was repeated three times, resulting in the formation of three repetitions, which served as the template for the construction of the transcriptome library.The procedure was then duplicated to procure two extra replicates.
Using the Trizol method, total RNA extraction was conducted [29].The prepared tissues were treated with Trizol reagent.After standing, they were centrifuged and mixed with chloroform.The aqueous layer was then separated and isopropanol was added to induce the formation of a precipitate.The quality and quantity of total RNA were weighed up by 1.5 % agarose gel electrophoresis and a microspectrophotometer.Quality control of RNA was performed by means of the Agilent 2100 bioanalyzer [30].The surplus RNA was carefully stored for subsequent quantitative real-time PCR validation.

Library construction and sequencing
The acquired total RNA was enriched for mRNA with polyA tails by Oligo (dt) magnetic beads.Subsequently, these isolated mRNAs underwent random fragmentation using divalent cations.This step laid the foundation for constructing a library, employing the standard library construction method from NEB.The first strand of cDNA was synthesized in the M-MuLV reverse transcriptase system using fragmented mRNA as a template and random oligonucleotides as primers, followed by degradation of the RNA strand with RNaseH and synthesis of the second strand of cDNA with dNTPs in the DNA polymerase I system.Following the purification of double-stranded cDNA, it underwent endrepair, A-tailing, and was connected to the sequencing junction.The cDNA fragments ranging from 250 to 300 bp were selected using AMPure XP beads.PCR amplification was carried out, and the resulting PCR products were purified once more using AMPure XP beads to ultimately obtain the library.The library construction kit was NEBNext® Ultra™ RNA Library Prep Kit for Illumina®.The Agilent 2100 Bio-Analyzer served to ascertain the insert size, which guaranteed the library's quality.In the final stage, the library was subjected to sequencing, which was executed on the Illumina NovaSeq 6000 platform.

Splicing and functional annotation of transcripts
Quality control analysis yielded clean reads, which were subsequently assembled via Trinity to produce reference sequences.The quality of the sequences was evaluated using BUSCO software [31].Upon obtaining spliced transcripts, gene function annotations were performed against six different databases: Swissprot, Nr, GO, KEGG, KOG/COG, and Nt.

Mapping and differential expression analysis
The Reference Sequence-based Estimation of Expression Model (RSEM) was utilized to align the purified reads of each specimen to the reference sequences.The number of reads that were mapped to each gene was designated as the read count.This read count was later converted to FPKM to approximate the levels of gene expression.DEGs were identified by employing DESeq2 software [32].The criteria used for identification included a q-value of less than or equal to 0.05 and an absolute value of log2 fold change greater than or equal to 1.

Function enrichment analysis
Functional enrichment analysis was performed on the DEGs.To determine the GO terms and the distribution of DEGs, GO analyses were deployed on the union set distinguished at two distinct time points.In addition, Gene Set Enrichment Analysis was employed to identify immune-related pathways and genes through the KEGG pathway analysis, thereby elucidating the functions of DEGs.Enrichment analyses of GO and KEGG were executed using the DAVID database (https://david.ncifcrf.gov/)2021 [33].

Construction of protein interaction network
Creating a PPI network can provide valuable insights into the relationships among immune pathways, facilitating the identification of key genes.In the present study, the STRING database (https://cn.string-db.org)was employed to construct a robust PPI network.

Validation of RNA-Seq
In the present study, 20 hub genes were highlighted for validation via qRT-PCR.The primary reference factor for identifying hub genes was the count of DEGs involved in interactions within the PPI network.This count was combined with the number of DEGs participating in the enriched KEGG signaling pathway.Utilizing Primer Premier 5.0 software, gene primer sequences were formulated based on the spliced transcriptome.Table 1 provides the sequence details for the 20 primers.The β-actin gene served as the reference due to its evident stability within the experiment.The fluorescence quantification methods implemented were adapted from Bao et al.'s research [34].

Sequencing and mapping
The sequencing process was administered for five distinct time points, each comprising three replicates namely, C0h_1, C0h_2, C0h_3, P6h_1, P6h_2, P6h_3, P24h_1, P24h_2, P24h_3, L6h_1, L6h_2, L6h_3, L24h_1, L24h_2, and L24h_3.The pertaining statistical data is presented in Table 2.An examination of the results indicates an average of 21,914,359 raw reads compared to 21,253,768 clean reads.The mean percentages for Q20 and Q30 were recorded as 96.52 % and 91.37 % respectively.The GC content exhibited an average of 38.57%, whereas the average mapping rate was duly noted to be over 70 %.

Screening of differential genes and expression analysis
Following LPS stimulation, a notable number of DEGs were detected, comprising 1236 upregulated and 793 downregulated genes within a 6hour timeframe.After 24 hours, the count decreased to 66 upregulated and 7 downregulated DEGs.The total number of genes displaying significant differential expression was 2099 (q-value ≤ 0.05, |Log2Fold-Change| ≥ 1) (Table S1-4).The distribution of such genes is clearly detailed in the volcano plots shown in Fig. 1A and 1B.Post-injection, an intersection of 3 genes was observed between the entire set of genes.The Venn Diagram shown in Fig. 1C demonstrates the overlap between the two-time frames.The focus of subsequent analysis was on the unified set of DEGs, taking into account both time points due to their potential integral role in immunological functions.The 2099 DEGs were further utilized for the hierarchical clustering heatmap analysis, and the differential gene expression profiles are depicted in Fig. 2.

GO and KEGG functional enrichment analyses
The task of performing GO functional and KEGG pathway enrichment analyses was carried out on a pool of 2099 DEGs.This GO annotation analysis yielded 657 level-3 subcategories within the Biological Process category, 173 under the Cellular Component category, and in the Molecular Function category.Level-3 is the third level of categorization of the GO database and enables a more detailed presentation of the specific functions undertaken by DEGs.Fig. 3 depicts the leading 20 Biological Process categories coupled with the top 10 subcategories from each aforementioned category.Illustrated in Fig. 4 is the level-2 KEGG diagram.As clearly showcased in Fig. 4, a significant portion of the DEGs were associated with the immune system.Subsequently, an analysis was conducted to identify 23 immune-related signaling pathways for a more comprehensive examination (Table 3).

PPI network analysis
The elaborately constructed protein association network is demonstrated in Fig. 5 which elucidates light on the intricate relationships between immune genes.Sixty DEGs involved in 23 signaling pathways closely related to the immune response were used to construct the PPI.Table 4 provides an in-depth description of the network's elements.

Verification of the RNA-seq accuracy using qRT-PCR
The validation of the 20 hub genes, as outlined in Table 5, was conducted through qRT-PCR.A comparative analysis was conducted between RNA-seq and qRT-PCR, utilizing fold change values.Fig. 6 demonstrates a consistent pattern in the observed expression trends between qRT-PCR and RNA-seq.

The clustering and enrichment analyses of DEGs
We first screened a set of DEGs based on transcriptomic data at the two investigated time points and performed a heatmap analysis using the concatenated set of DEGs (2099) at these two time points.Heat map analysis showed significant clustering and expression pattern differences between the AL group and the AP group, which further validated the significant effect of LPS stimulation on gene expression patterns.In addition, although there were small differences between theAP and AB groups, they may be related to the effect of the injection stimulus on the immune response.GO and KEGG functional enrichment analyses further confirmed that immune responses were significantly activated in A. fangsiao after LPS stimulation.For example, a large number of GO terms related to the immune response were enriched, including the Wnt signaling pathway, endocytosis, positive regulation of vascular endothelial cell proliferation and leukocyte migration, were enriched in biological processes [35][36][37].Similarly, various immune-associated KEGG signaling pathways, including chemokine signaling pathway, Hippo signaling pathway, and leukocyte transendothelial migration were identified in the KEGG level-3 subclass [38][39][40].However, a gradual decrease in the number of significantly DEGs was observed over time, possibly suggesting that the intensity of LPS stimulation was too high, leading to a failure of immune defenses.In order to further expound the mechanisms of leucosomal immunity to LPS stimulation in A. fangsiao, an in-depth exploration of the functions of these genes, as well as the effects of LPS stimulation on immune defense functions, is necessary.

Identification of the hub genes
Proteins, as primary facilitators of diverse biological processes, regulate life processes such as metabolism, signal transmission, cell cycle, and gene expression [41,42].In the present study, a protein interaction network was constructed based on 60 DEGs derived from immune-related pathways.We found that the proteins in this network exhibit a higher degree of interconnectivity with each other compared to a randomly selected set of similar size, which further confirms the biological importance and relevance of the proteins we studied.We hypothesize that the hub proteins in these networks may have a crucial role in immune function because of their high number of interactions in the network.Therefore, we defined proteins with a high number of interactions as hub proteins and designated their corresponding genes as hub genes.

Discussion of hub genes and pathways
Through PPI analysis, we successfully predicted and screened 20 hub genes with multiple interactions that play important roles in immune regulation.These genes were categorized into eight specific functional groups encompassing multiple immune-related pathways, including the Caspase family, the Hippo signaling pathway, the Fc epsilon RI signaling pathway, the Chemokine signaling pathway, Inflammatory mediator regulation of TRP channels, Natural killer cell-mediated cytotoxicity, Leukocyte transendothelial migration, and the VEGF signaling pathway.

Caspase family
The essential role of apoptosis, also referred to as programmed cell death, in development and maintaining tissue homeostasis is of paramount importance [43,44].Serving a central function in apoptosis and inflammatory response management is the caspase family, which facilitates the elimination of infected cells, the maintenance of internal environmental stability, and immunity enhancement [45][46][47][48].As is well-documented, caspase family genes, such as CASP1 [49], CASP4 [50], CASP5, and CASP11 [51], are influential in immune regulation.In the present research, CASP3, CASP7, and CASP8 as key genes involved in significant immune processes in the A. fangsiao species.CASP3, in particular, plays a pivotal role in the execution phase of programmed cell death and has recently been recognized as a mediator of inflammation [52].CASP7 is linked closely with immune cells such as neutrophils and dendritic cells [53], playing a significant role in apoptosis [54].Similarly, CASP8 regulates pyroptosis, necroptosis, and apoptosis as a molecular checkpoint, mitigating tissue damage by managing inflammatory response [55].An analysis of their expression levels revealed a significant upregulation at the six-hour time point, followed by a decline at the 24-hour mark.We speculate that the dynamic expression of CASP3, CASP7, and CASP8 genes after LPS stimulation may reflect their different stages of roles in the A. fangsiao immune response.The upregulation at 6 h may be a rapid response after inflammatory infection to clear infected cells and maintain immune homeostasis by regulating apoptosis and inflammatory responses.In contrast, the marked decline after 24 h may reflect a gradual waning of the inflammatory stimulus and a regulatory and recovery phase of the immune response.This is an important manifestation of the stress immune response of A. fangsiao against bacterial infection.

Hippo signaling pathway
The Hippo signaling pathway has been demonstrated to be crucial in maintaining homoeostasis of the immune system and regulating the size of immune organs [56].Such pathway regulates apoptosis, cellular proliferation, and stem cell self-renewal, impacting tissue homoeostasis, organ size, and tissue remodeling [57].Further, recent research has demonstrated the Hippo pathway's role in regulating innate immunity [58].During the present investigation, findings were made that several critical genes -PRKCI, YAP1, and PPP1CA were enriched in this pathway.PRKCI is involved in activating NF-kappa-B and provides a broad protective effect against apoptotic stimuli [59,60].Such mechanism aids in damage protection by mitigating excessive immune responses.YAP1 activates the transcription of pro-inflammatory cytokines, which recruit and expand immune cells.Moreover, studies have suggested that PRKCI promotes immune suppression via YAP1 [61].PRKCI is one of three catalytic sub-units for protein phosphatase 1, which negatively regulates RIG-I-like receptors and Toll-like receptors to balance the immune system [62].In the present study, these three critical genes exhibited significant up-regulation at six hours, with a decrease observed at twenty-four hours.We hypothesized that they maintain immune system homeostasis by altering transcript expression levels in response to LPS stimulation.For example, the immune function of A. fangsiao white body after bacterial stimulation is enhanced by mechanisms such as anti-apoptosis, anti-injury, and receptor control.

Chemokine signaling pathway and Leukocyte transendothelial migration
Chemokines, a sub-group of small cytokines, aid the recruitment of various leukocyte subsets and direct leukocyte migration to definitive locations during inflammatory responses.In parallel, they are indispensable for facilitating interactions among immune cells [63,64].The transendothelial migration of leukocytes, essential for triggering both innate and adaptive immune responses, considerably impacts immune surveillance across organisms [40,65].In our study, we observed that LPS stimulation may be mediated by activation of chemokine signaling pathways and leukocyte transendothelial migration signaling pathways.This may lead to the release of cellular signals and factors that trigger the migration of immune cells such as leukocytes and macrophages toward the site of infection or injury.At the same time, the synergistic effect of these processes helps accelerate the localization and aggregation of immune cells at the site of infection or injury.This in turn accelerates the initiation of the inflammatory response and the clearance of the foci of infection.Furthermore, Phospholipase C Gamma 1 (PLCG1) was identified as a central gene enriched in these two signaling pathways.Acting as an information conduit, PLCG1 controls intracellular signaling cascades, actin reorganization, and cellular migration [66].A significant upregulation of this gene was observed at 6 hours, which subsequently declined at 24 hours.This fluctuating expression pattern significantly enhanced immune cell migration and communication, thereby strengthening resistance to bacterial invasion in the white body tissue of A. fangsiao.In summary, the Chemokine signaling pathway and Leukocyte transendothelial migration was critical to the immune system of A. fangsiao white body.However, the specific molecular mechanism and biological effects still need to be confirmed by further experimental studies.

Top three hub genes
In the present research, protein interactions were examined to determine hub genes, and AKT3, MAPK14, and PIK3CA were ultimately identified as the most predominant based on strong protein-protein interactions.AKT3, from the serine/threonine kinase family, significantly impacts various biological regulation processes [67].Notably, it modulates cell proliferation, and inhibiting AKT3 leads to cell cycle cessation in the G1 phase [68].Moreover, AKT3 is associated with the viability of immune cell types such as monocytes and macrophages [69].The MAPK14 gene, a member of the MAP kinase family, serves as an innate immune system modulator, particularly activating macrophages.Similarly, MAPK14 plays a vital role in immune response cascades triggered by external stimulation, such as physical stress and inflammatory cytokines, and directly activates transcription factors [70][71][72][73].PIK3CA, an essential component of the PI3K signaling pathway, recruits proteins that possess pH domains to the cell membrane, initiating the signaling cascade responsible for processes such as proliferation, cell growth, and survival [74,75].The PI3K/AKT signaling pathway crucially regulates various cellular processes [76,77].In the present study, AKT3 and PIK3CA were identified as hub genes with significant 6 h upregulation, indicating their critical immune function via the PI3K/AKT cascade pathway.For example, it can regulate cell proliferation and growth, and participate in the PI3K/AKT signaling pathway to regulate immune cell activation and promote the initiation of immune response.At the same time, it regulates cell survival and apoptosis and helps maintain immune homeostasis and tissue homeostasis.MAPK14 also exhibited significant upregulation at 6 hours, followed by a decrease at 24 hours.This underscores the importance of these top hub genes in A. fangsiao's immune responses.Specifically, processes such as cell proliferation, pro-inflammatory responses, and immune cell activation play significant roles in A. fangsiao's white bodies.This underscores the need for future in-depth investigations to elucidate their specific mechanisms.

Other hub genes and pathways
The observed results encompass several pathways and genes that have not been previously mentioned.These include the Fc gamma Rmediated phagocytosis, the regulation of TRP channels by inflammatory mediators, the VEGF signaling pathway, cytotoxicity mediated by natural killer cells, and the involvement of NOTCH2, RAC1, and SMAD4 genes.Fc gamma R-mediated phagocytosis are classical inflammatory or immune-related signaling pathways that can be activated by LPS stimulation [78].TRP channels are ion channels on cell membranes that play an important role in immune regulation.Modulation of TRP channels by inflammatory mediators may affect intracellular calcium ion concentration and cellular activation, which are involved in the regulation and transmission of inflammatory responses [79].VEGF is involved in the regulation of inflammation and immune responses by modulating angiogenesis and permeability, as well as promoting migration and activation of immune cells [80].Natural killer cells are important components of the immune system that recognize and kill infected and tumor cells [81].We hypothesize that after LPS stimulation, A. fangsiao leukosomes may act synergistically through Fc gamma R-mediated phagocytosis and cytotoxicity mediated by natural killer cells to jointly

Conclusion
Transcriptome-based protein-protein interaction analysis reveals the complexity and diversity of the A. fangsiao immune gene network.Our study confirms the significant effect of LPS stimulation on the immune mechanism of white body of A. fangsiao, further revealing the key pathways and genes involved in the infection and immune regulation process.In particular, we identified several important immune-related pathways, such as chemokine signaling pathway, Hippo signaling pathway, and Leukocyte transendothelial migration, as well as significant expression changes of hub genes, such as AKT3, MAPK14, and PIK3CA.These findings deepen our understanding of the immune mechanisms of A. fangsiao and provide new ideas and targets for future research and immunotherapy.

Declaration of competing interest
The authors declare that they have no known competing financial  interests or personal relationships that could have appeared to influence the work reported in this paper.

Fig. 1 .
Fig. 1.Distribution and number of DEGs.(A) The volcano plot of differential gene expression measured at 6 hours.The red dots denote upregulated DEGs, the green dots denote downregulated DEGs, and the blue dots denote genes having no differential expression.(B) The volcano plot of differential gene expression captured at 24 hours.(C) Venn diagrams for DEGs.The yellow areas indicate the number of DEGs significantly differentially expressed at 6 h, the purple areas indicate the number of DEGs significantly differentially expressed at 24 h, and DEGs significantly differentially expressed at all time points are shown in dark red.

Fig. 2 .
Fig. 2. Hierarchical clustering heatmap of the DEGs.The x-axis signifies the distinct time points, each denoted by a unique horizontal line representing an individual gene.A color gradient extending from red to green indicates a transition from high to low expression levels of DEGs, respectively.

Fig. 3 .
Fig. 3.The GO enrichment analysis of DEGs.The X-axis illustrates the GO terms, and the Y-axis signifies the quantity of DEGs.Each category is represented by different colored bars: the biological process is shown in orange, the cellular component in blue, and the molecular function appears in magenta.

Fig. 4 .
Fig. 4. Distribution of level-2 KEGG classes.The horizontal axis denotes the count of DEGs, while the vertical axis signifies specific level-2 KEGG classes.Level-2 is the second level of categorization of the KEGG database, which enables a more systematic presentation of the biological processes in which DEGs are involved.
clear infected cells and pathogenic microbes.Regulation of TRP channels by inflammatory mediators may affect vascular endothelial cell function and vascular endothelial growth factor signaling pathway activity.This may influence the migration of immune cells and the development of tissue inflammation.Proteins encoded by the NOTCH2 play a key role in cell signaling and are involved in the regulation of cell proliferation, differentiation and fate decisions[82].Rac1 belongs to the Rho GTPase family .It acts as an intracellular signal transducer involved in the regulation of various biological processes such as cytoskeletal organization, gene transcription and cell proliferation[83].SMAD4 is a key factor regulating TGF-β signaling and plays an important role in regulating biological processes such as cell proliferation, differentiation and apoptosis[84].In our study, differential expression of NOTCH2, Rac1 and SMAD4 were all observed to be up-regulated after 6 h of LPS stimulation of A. fangsiao white body.This suggests that NOTCH2, Rac1 and SMAD4 may synergistically regulate immune cell migration, cell proliferation and inflammatory responses to attenuate the infectious effects of LPS.The focus of future research should be on further exploring the immunological functions these components serve in the white tissue of A. fangsiao.

Fig. 5 .
Fig.5.The immune-related protein interaction network.Each node represents a protein, and the lines between the nodes represent that they have an interaction relationship.The number of lines between the nodes represents the strength of the interaction.

Fig. 6 .
Fig. 6.Comparison of the 20 hub DEGs between qRT-PCR and RNA-seq expression levels.The horizontal axis signifies the time point post Lipopolysaccharide (LPS) stimulation, and the vertical axis illustrates the fold change.The same trend of change at different time points represents that the RNA-Seq results are accurate.

Table 1
List of primers used for quantitative RT-PCR validation.

Table 2
List of sequencing information.

Table 3
A summary of 23 immune pathways.

Table 4 A
summary of the PPI network.
Z. Lu et al.

Table 5
Statistics of 20 hub genes.