β-Conglycinin regulates distal intestinal immunity through the CIITA-mediated MHC II-PI3K/Akt/mTOR signaling pathway in hybrid grouper (Epinephelus fuscoguttatus (cid:0) × E. lanceolatus (cid:0) ): The ameliorative effects of sodium butyrate (NaB)

β-Conglycinin (7S) a strong protein in the of whereas of sh. In this study, we investigated the effects of low and high doses of 7S and the ameliorative effects of NaB (based on high-dose 7S) on the growth performance, serum immunity, distal histopathology, and CIITA-mediated MHC II-PI3K/Akt/mTOR pathway in hybrid groupers (Epinephelus fuscoguttatus (cid:0) × E. lanceolatus (cid:0) ). The results revealed that the specic growth rate of groupers signicantly increased, decreased, and increased in the low-level 7S (bL), high-level 7S (bH) high-level 7S plus NaB (bH-NaB) groups, respectively. The feed coecient ratio was signicantly increased in the bH and bH-NaB groups, whereas serum levels of IgE, IFN-γ, IL-1β, and TNF-α were upregulated in the bH group, and IgE was upregulated in the bH-NaB group. With respect to distal intestine histopathology, the intestinal diameter/plica height ratio was signicantly increased in the bH group. Furthermore, there were increases in nitric oxide, nitric oxide synthase (NOS), and peroxynitrite anion (ONOO) in the bH group, and decreases in NOS and ONOO in the bH-NaB group In the distal intestinal tract, the mRNA levels of TSC1, mTOR C2, CIITA, and CREB1 were signicantly upregulated in all three treatment groups, whereas those of IKKα, Rheb, mTOR C1, mLST8, EIF4B, NFY, GILT, and AEP were upregulated and downregulated in the bH and bH-NaB binding protein p70 S6K, ribosomal protein kinase EIF4E, translation initiation factor 4E; EIF4B, translation initiation factor 4B; S6, small subunit ribosomal protein target of rapamycin complex subunit; rapamycin-insensitive companion of proline-rich protein Ras homolog gene family, member PKCα, protein kinase histocompatibility complex class II trans-activator; RFX5, cyclic AMP-responsive element-binding protein nuclear transcription factor histocompatibility complex class I histocompatibility class II gamma-interferon-inducible-lysosomal reductase; asparaginyl endopeptidase;


Background
The hybrid grouper (Epinephelus fuscoguttatus × E. lanceolatus ) is characterized by a rapid growth rate and desirable meat quality, which is of high economic value (Wu, et al., 2017). As a typical carnivorous marine sh, hybrid grouper typically require up to 50-55% protein in their feed (Jiang, et al., 2016), which is generally provided by shmeal containing high levels of nitrogen and phosphorus In this study, we therefore used hybrid groupers as experimental animals to evaluate the hypothesis that dietary β-conglycinin might affect growth by regulating serum immunity, distal intestinal histopathology, epithelial injury, and the PI3K/Akt/mTOR signaling pathway. In addition, we also investigated the potential protective effects of appropriate doses of NaB against high levels of dietary 7S. We anticipate that the ndings of this study will provide a reference for research on improving the tolerance of hybrid grouper to soybean meal and reveal the mechanisms underlying the protective effects of NaB on the distal intestine.

Animals
The juvenile hybrid grouper used in this study were purchased from a commercial hatchery on East Island (Zhanjiang, China), and were derived from the same batch of arti cially hatched sh. Prior to experimentation, the sh were acclimated to the local environment by placing in an outdoor pond of the Biological Research Base of Guangdong Ocean University for 1 week, during which time they were provisioned with commercial feed.

Feeding trial and challenge test
Having acclimated, 480 healthy uniformly sized hybrid groupers were selected and randomly divided into four groups, each with four replicates of 30 sh, and the sh were cultured in 300-L glass ber buckets. During the culture period, the sh were fed twice daily (at 08:00 and 16:00) to apparent satiety with a water change of approximately 70%. Dissolved oxygen levels in the containers were maintained at ≥7.00 mg/L via continuous aeration using air stones, whereas water temperature, pH, and ammonia nitrogen content were maintained at 29.00 ± 1.30°C, 7.8-8.1, and <0.09 mg/L, respectively. At the end of the 8week culture period, 40 sh per treatment were randomly selected for a challenge test (10 sh per replicate). Each sh was injected intraperitoneally with 200 µL of Vibrio parahaemolyticus at a concentration of 7.41 × 10 8 CFU/ml, and thereafter the sh were continuously observed for 1 week, with mortality being recorded daily.

Diet formulations
Experimental sh were provided with one four diets containing protein and fat at levels of approximately 48% and 10%, respectively, which were prepared using red sh meal, casein, and gelatin as the main protein sources, and sh oil and soy lecithin as the main lipid sources. As treatments, the diet was supplanted as follows: 0% 7S, 0% NaB ( shmeal group, FM); 1.50% 7S (low-level 7S, bL), 0% NaB; 6.00% 7S, 0% NaB (high-level 7S, bH); 6.00% 7S, 0.13% NaB (high-level 7S with NaB, bH-NaB) ( Table S1). The three experimental diets were also supplemented with methionine and lysine to same level as that in the FM diet. The essential amino acid pro les of the diets used in this experiment are shown in Table S2. Puri ed 7S was purchased from the China Agricultural University (Patent No. 200410029589.4, China).
For use in the experimental diets, water was added to the puri ed β-conglycinin several times in small amounts, stirred thoroughly, and the pH of the solution was adjusted to 7.2. The 7S solution was then placed in a glass petri dish and dried for 72 h in a low-temperature freeze dryer. After crushing the different raw materials of the diet through 380-μm meshes, the components mixed in a stepwise manner, adding sh oil, soybean lecithin, and 30% water and mixing evenly. The resulting mixture was then placed into a feed pelletizer, which produced feed pellets of 2.5 mm in diameter. The pellets were air-dried at room temperature until the moisture content was approximately 10% (approx. 48 h), sealed in a selfsealing bag, and stored at -20°C until required. The 30% microencapsulated NaB used in this study was kindly provided by Shanghai Menon Animal Nutrition Technology Co., Ltd.

Sample collection
After the end of the 8-week breeding experiment, the sh in each bucket were fasted for 24 h and then counted and weighed. For each replicate, two sh were randomly selected, from which blood was drawn using a 1 mL syringe. After being left to stand for 12 h, the blood samples were centrifuged for 10 min at 5000 g/min and 4°C. The serum thus obtained was collected and stored at -80°C for the determination of serum immune indicators. A further six sh were randomly selected for each replicate, from which the intestines were gently removed, and the segment of intestine near the cloacal hole was cut as the distal intestine (DI). Two of the DI segments for each replicate were placed in 4% formaldehyde solution for the preparation of Alcian Blue-Periodic acid-Shiff (AB-PAS) stained intestinal sections, and a further two DI segments were placed in liquid nitrogen for the determination of hindgut-related enzyme activities. After the DI tissue had been thawed on ice, dried with qualitative lter paper, and weighed, a sample was homogenized in homogenization medium (0.8% sodium chloride solution) in an ice bath at a sample: medium ratio of 1:9 (mass to volume ratio), centrifuged at 5000 g/min for 10 min at 4°C, and the supernatant was collected for analysis. The remaining two DI samples from each replicate were placed in RNA latter, and after being left to stand for 12 h, were stored in a freezer at -80°C for subsequent determination of related gene expression.

Determination of growth and DI evaluation indicators
Parameters of interest were determined as follows: WGR (weight gain rate, %) = 100 × ( nal weight -initial weight)/initial weight SGR (speci c growth rate, %) = 100 × [ln( nal weight) -ln(initial weight)]/days of experiment SR (survival rate, %) = 100 × nal sh number/initial sh number FCR (feed coe cient ratio) = dry feed consumed/weight gain Id/Ph = intestinal diameter/plica height Immune index determinations Serum immune and DI injury indices were determined using Shanghai Enzyme-Linked ELISA immunoassay kits (Full-wavelength microplate reader-1510). AB-PAS sections of DI were prepared by Wuhan Servicebio Biological Technology Co., Ltd. Observations were made using a Leica DM 6000 optical microscope, and measurements and photographs were taken using cellSens Standard 1.8 and LAS 3.8 software, respectively, with 10 randomly selected plicae and muscle layers being measured in each section.
Total RNA from hybrid grouper DI was extracted using a TranZol Up Plus RNA kit (Beijing TransGen Biotechnology Co., Ltd) in accordance with the manufacturer's instructions. The integrity of the extracted RNA was assessed by 1% agarose gel electrophoresis, and a NanoDrop ND-2000 spectrophotometer (Thermo Scienti c, USA) was used to determine the purity and concentration of RNA. RNA samples of su cient quality should run as a clear and complete band, and have an OD 260 /OD 280 ratio of between 1.8 and 2.0. A PrimerScript TM RT-PCR Kit (TaKaRa, Kusatsu, Japan) was used to reverse transcribe total RNA to cDNA, which was stored at -20°C prior to RT-PCR analysis. Speci c primers for RT-PCR (Table S3) were designed using Primer Premier 5.0 software based on full-length transcriptome sequences of hybrid grouper in the NCBI Sequence Read Archive with accession number PRJNA664416. All the real-time PCR reactions were run in an Applied Biosystems 7500 Real-Time PCR System (Life Technologies, Carlsbad, CA, USA) using a SYBR ® Premix Ex Taq TM Kit (Takara). The β-actin gene was used as an internal reference to normalize the ampli cation e ciency of each target gene, and the mRNA expression levels of genes were compared and analyzed using 2 -ΔΔCT methods according to Livak et al. (Livak, Schmittgen, 2001).

Statistical analyses
All data were subjected to one-way analysis of variance followed by Tukey multiple range tests to determine signi cant differences among treatment groups using SPSS v. 22 (IBM, USA) at a signi cance level of P < 0.05, as described by Guo et al. (Guo, et al., 2017). The results are presented as the means ± SE.

Growth performance and challenge test
The results obtained for the growth performance of sh in different groups are presented in Table 1.
These results indicated that compared with the FM group, the nal body weight (FBW), weight gain rate (WGR), and speci c growth rate (SGR) were all signi cantly higher in the bL group and lower in the bH group, whereas no signi cant differences were observed between the FM and bH-NaB groups. The feed coe cient ratio (FCR) in the bH group was found to differ signi cantly from that in the other three groups, although there was no signi cant difference between the FM and bL groups. In contrast, we detected no signi cant differences among the four groups with respect to survival rate (SR).
The cumulative mortality of sh in response to injecting with V. parahaemolyticus is shown in Figure S1, which indicates that sh in the bH and bH-NaB groups suffered heavier mortalities than those in the FM and bL groups, with all groups reaching an asymptote at approximately 5 days post-injection. Among the different groups, the bH group showed a sustained and rapid increase in cumulative mortality from the rst day post-injection, whereas in the bH group, mortality increased slowly over the rst 3 days after injection, although thereafter showed a rapid increase.

Serum immune indices
The serum immune indices determined for the different groups are presented in Figure. 1. Compared with the FM group, we detected signi cant increases in serum IFN-γ in the bL, bH, and bH-NaB groups, among which the bH-NaB group showed the most signi cant difference. There were no signi cant difference between the FM and bL groups with respect to IgE levels, and the same trend was observed between the bH and bH-NaB groups, whereas IgE levels in these two groups were found to be signi cantly upregulated compared with the FM and bL groups. Furthermore, the levels of IL-1β were found to be signi cantly increased in the bH group and signi cantly reduced in the bL and bH-NaB groups, although no signi cant differences were detected between the bL and bH-NaB groups. With respect to TNF-α, we observed signi cantly downregulated and upregulated levels in the bL and bH groups, respectively, compared with the FM group, whereas in contrast, there was no signi cant difference between the bH-NaB and FM groups.

Distal intestinal histopathological evaluation and damage indicators
The results obtained for the different groups with respect to distal intestinal histopathology evaluated by AB-PAS staining, developmental status assessment, and damage indicators determined by ELISA are presented in Figures 2, 3, and 4, respectively. As shown in Figure 3, the intestinal diameter/plica height ratios (Id/Ph) obtained for the bL and bH-NaB groups were signi cantly higher than that for the FM group, and signi cantly lower than that for the bH group. Among the four groups, the bH group was found to have the lowest density of mucus cells, whereas we detected no signi cant differences among the other three groups.
Compared with the FM group, the levels of nitric oxide (NO) were signi cantly upregulated in the bH and bH-NaB groups (Figure 4). Similarly, although the levels of NO in the bL groups were somewhat lower, they were still signi cantly higher than those in the FM group. Whereas we observed that levels of nitric oxide synthase (NOS) in the bH group were signi cantly higher than those in the FM, bL, and bH-NaB groups, we detected no signi cant difference among the FM, bL, and bH-NaB groups. Moreover, levels of the peroxynitrite anion (ONOO) were found to be lowest in the bL group, whereas no signi cant differences were observed among the FM, bH, and bH-NaB groups.

Distal intestinal immune-related gene expression andproteinexpression
The effects of 7S and NaB on the distal intestine of hybrid grouper are presented in Figure 5. Compared with the FM group, the mRNA levels of PI3K, RS5, PRAS40, p70 S6K, and PRR5 were upregulated in the bL and bH groups, and were signi cantly upregulated in the bH-NaB group. Similarly, we observed that the mRNA levels of 3-PDK1, mTOR C1, TEL2, EIF4E, EIF4B, Sin1, NFY, MHC I, GILT, AEP, and TCR were all upregulated in the bL and bH-NaB groups, and were signi cantly upregulated in the bH group. Although Rheb, Raptor, mLST8, and 4EBP1 mRNA levels were also signi cantly upregulated in the bH group, no changes were detected between the bL and bH-NaB groups and the FM group.
In addition, the bL, bH and bH-NaB groups all showed signi cantly higher levels of TSC1, mTOR C2, 4EBP1, CIITA and CREB1 mRNA compared with the FM group, whereas no signi cant differences were detected between the bH and bH-NaB groups. Although we found that the mRNA levels of deptor, RICTOR, SGK1, RFX5, and MHC II in the bH and bH-NaB group were signi cantly higher than those in the FM and bL groups, no signi cant differences were observed between the FM and bL groups or between the bH and bH-NaB groups. PKC mRNA levels were upregulated in the bH-NaB group and downregulated in the bL group, and those of RhoA were upregulated in the bL and bH-NaB groups compared with the FM group. Moreover, the mRNA expression of CD4 was signi cantly downregulated in the bL, bH, and bH-NaB groups, whereas we detected no signi cant differences among the four groups with respect to TSC2 and S6.

Discussion
Growth performance and challenge test In general, due to the incomplete development of the immune system in juvenile animals, the intestines of individuals often show an allergic reaction to 7S, resulting in metabolic disorders and adverse physiological and biochemical reactions (Han, et al., 2018), which can have the effect of disrupting the digestion and absorption of nutrients in the intestines . Interestingly, a low dose of 7S can often induce a response similar to that obtained with immunostimulants (Zhou, et al., 2015), which is often manifested in terms of modi ed growth performance. For example, in turbot (Scophthalmus maximus L.), a reduction in feed utilization e ciency following ingestion of feeds containing 4%-8% 7S has been observed to result in a signi cant reduction in speci c growth rate (SGR) (Li, et al., 2017b). These ndings were similar to those obtained in the present study, in which we observed that hybrid grouper receiving a diet containing 8% 7S showed a signi cant reduction in SGR and weight gain (WG). However, whereas in turbot, the effect of ingesting of a diet containing 2% 7S did not differ signi cantly from that of a control diet with respect to SGR, we observed an increase in SGR in hybrid grouper fed a diet supplemented with low-dose 7S. Moreover, in turbot, it was found that the level of soybean supplantation and WG showed a rst-degree relationship (Gu, et al., 2016). Hybrid groupers can, however, tolerate up to 30%-50% replacement of shmeal with soybean meal in their feed, and experience no adverse effects on growth, even at soybean meal substitution levels of 10% and 30%, WG increased while did not show a signi cant difference (He, et al., 2020). These observations thus indicate that hybrid grouper may be more tolerant to soybean meal than are turbot, and therefore we speculate that for turbot, the addition of 2% soybean globulin is higher than the dose used for immune enhancement.
In the present study, we found that the addition of sodium butyrate (NaB) signi cantly increased SGR in the bH-NaB group compared with that in the bH group, indicating that NaB can be effective in ameliorating the growth inhibition effects attributable to high doses of 7S. We also found that the feed coe cient ratio of sh in the bH and bH-NaB groups was signi cantly higher than that in the FM and bL groups, whereas the addition of NaB signi cantly reduced the FCR, indicating that NaB may enhance the growth performance by improving the feed utilization of hybrid grouper, which is similar to results obtained for grass carp Liu, et al., 2014). We speculate that this effect of NaB is associated with an increase intestinal antioxidant capacity and enhancement of intestinal immunity (Mirghaed, et al., 2019).
To further investigate the effects of 7S and NaB on the disease resistance of the hybrid grouper, we conducted a 7-day challenge test using Vibrio parahaemolyticus injected into sh at a concentration of 7.41 × 10 8 CFU/mL at the end of the breeding experiments. We accordingly observed the highest cumulative mortality in the bH group, thereby indicating that high doses of 7S can signi cantly reduce disease resistance in hybrid groupers, whereas resistance was enhanced to some extent in groupers fed a diet supplemented with NaB. Interestingly, whereas the trend in mortality in the bH group showed a consistent and rapid increase during the rst 4 days post-injection, the upward trend only became apparent at day 4 post-injection in groupers fed the NaB-supplemented diet. These observations thus tend to indicate that although NaB may not be effective in ameliorating the immunosuppression caused by high doses of 7S, it can, to a certain extent, delay the onset of disease caused by V. parahaemolyticus, thereby gaining valuable time for treatment. However, given that it was unclear how this resistance is regulated, we conducted the research described in the following section.

Serum immunity
It is widely believed that the allergic responses to speci c antibody-mediated humoral immunity and Tcell-mediated cellular immunity, which induce the production of serum-speci c antibodies, occur primarily in the intestinal tract of sh ingesting 7S (Mclean, Ash, 1986;O"Donnell, et al., 1996). In this regard, cytokines play an important role in regulating immune responses and the functions of IgE as an important mediator of allergic responses. As an antigenic protein, 7S is processed by antigen-presenting cells and is bound by IgE antibodies produced by plasma cells (Krishnan, et al., 2009;Zheng, et al., 2012). When the antigen re-enters the body, binding to antibodies causes mast cells to release allergic substances such as histamine, thereby inducing an adverse reaction (Amigo-Benavent, et al., 2011; Ogawa, et al., 1995). With respect to the ndings of the present study, we found that low-dose 7S did not appear to stimulate an increase in IgE production in hybrid grouper, whereas in contrast, supplementation with high-dose 7S signi cantly increased the levels of IgE, thereby indicating that ingestion of high doses of 7S may cause intestinal damage by activating B cells or T cells, which thus produce higher amounts of IgE, and consequently generate higher levels of toxic substances (Sun, et al., 2013). Similar results have also been obtained in piglets (Hao, et al., 2009b), and in several previous studies, NaB has been shown to enhance IL-4-induced IgE production (Yamamoto, et al., 1996), and can also improve clinical symptoms and nasal mucosal epithelial morphology by reducing serum IgE levels in mice (Wang, et al., 2016). Thus, on the basis of the ndings of previous studies and the study reported herein, it would appear that NaB does not alleviate high-dose 7S-induced allergic reactions by lowering serum IgE.
As a typical pro-in ammatory cytokine, IL-1β is primarily produced by mononuclear macrophages, and can induce the activation of neutrophils and macrophages, stimulate the release of thromboxane and platelet-activating factor, increase the permeability of epithelial and endothelial cells, and induce intestinal in ammation (Al-Sadi, et al., 2012). In the present study, we found that levels of IL-1β in bL and bH group sh were signi cantly down-and upregulated, respectively. The lower levels of IL-1β in the bL group conceivably indicate that low-dose 7S can maintain normal serum immune responses by modifying IL-1β secretion, whereas the signi cantly elevated levels of IL-1β in the bH group, could indicate that at higher doses, 7S may promote CD4 + T-cell activation, proliferation, and differentiation by enhancing the abnormal secretion of IL-1β, thereby exacerbating the in ammatory response (Sun, et al., 2013). We also observed that NaB supplementation of the grouper diet had the effect of signi cantly reducing serum IL-1β, thereby indicating that NaB may modulate serum immunity by inhibiting the abnormal secretion of IL-1β. In this regard, studies on rats have found that NaB can induce the overexpression of IL-1β by inhibiting the binding of p65 to the IL-1β promoter (Zhong, et al., 2017). We accordingly speculate that a similar mechanism operates in the hybrid grouper.
TNF-α is a further key cytokine that plays role in mediating intestinal in ammatory processes (Parker, et al., 2019), and has been shown to exacerbate the progression of enteritis by inducing the release of proin ammatory factors (Lang, et al., 2004). Consequently, determining changes in the levels of TNF-α is important with respect to monitoring in ammatory responses in the body. We found that a low dose of 7S signi cantly reduced serum TNF-α levels in the hybrid grouper, whereas in contrast, a high dose had the effect of promoting the secretion of TNF-α. Similarly, in turbot, it has been found that supplementing feed with 4% to 8% 7S increased the expression of TNF-α, which ultimately inhibited growth (Li, et al., 2017b). These observations indicate that 7S has a regulatory effect on TNF-α secretion in both hybrid grouper and other sh. Indeed, it has previously been found that the percentage of CD4 + lymphocyte subpopulations in mice increases linearly with increasing 7S levels, and thus we hypothesized that the elevation of TNF-α in the high-dose 7S group may be associated with an increase in CD4 + lymphocyte sub-populations in groupers (Fei, et al.). We also found that supplementation of the grouper diet with NaB had the effect of reducing serum TNF-α to levels comparable with those observed in FM group sh, which we suspect could be attributable to the fact that NaB can inhibit TNF-α secretion by down-regulating LITAF expression (Han, et al., 2018). This effect may also be associated with the fact that NaB inhibits mast cell activation and suppresses the release of mast cell in ammatory mediators (Assem, et al., 2008;Wang, et al., 2018).
Similarly to TNF-α, the type II interferon IFN-γ play roles in immune and in ammatory responses. 7S is treated by APC cells and the 7S soymetide is exposed, which binds to B-or T-cell-transformed immunoglobulins (Tsuruki, et al., 2003). On the basis of the ndings of the present study, it would appear that both low and high doses of 7S can promote the secretion of IFN-γ, and we accordingly speculate that this may be closely related to the activation of T cells by 7S as an antigenic protein. This assumption tends to be supported by our observations indicating that dietary supplementation with NaB was associated with a signi cant elevation in the levels of serum IFN-γ. NaB was also found to inhibit certain IFN-γ functions by inhibiting the downstream signaling of IFN-γ and thus the production of IFN-γ-induced factors (Kitamura, et al., 2010). We accordingly conjecture that, similar to humans, NaB can reduce the production of toxic substances in groupers by blocking the induction of IFN-γ on in ammatory cells. To examine this possibility, we conducted the experiments described in the following section.

Distal intestinal damage and histopathology
Nitric oxide (NO), a free radical gas produced from l-arginine via the catalytic activity of nitric oxide synthase (NOS), plays an important role in host defense and in ammatory responses, and can interact with cytokines such as TNF-α and IFN-γ, thereby in uencing the course of in ammatory responses (Kim, et al., 2019;Kubes, Mccafferty, 2000;Nakanishi, et al., 2018). Currently, however, there is a degree of controversy as to the mechanism whereby NO is regulated in the intestines (Anand, et al., 2006;Kubes, Mccafferty, 2000). In the present study, we found that NOS showed an elevated trend in the bL group and NO was signi cantly elevated in the sh in this group. A similar pattern was also observed in groupers in the bH group. Notably in this regard, we also detected signi cant increases and decreases in the levels of IFN-γ and TNF-α in the bL group, respectively, the former of which can enhance NO secretion by stimulating macrophages and inducing the production of nitric oxide synthase (Nieto-Patlán, et al., 2019). However, given that TNF-α showed an increase and decrease in the bL and bH groups, respectively, we were unable to determine whether TNF-α is involved in the regulation of NO production. Nevertheless, we speculate that either TNF-α is not involved at low doses of 7S but is involved at high doses, or, alternatively, that TNF-α does not play a role in regulating the macrophage production of NO in the hybrid grouper. In addition, we observed that dietary supplementation with NaB maintained the signi cant upregulated production of IFN-γ, and resulted in the recovery of TNF-α to FM group levels. Correspondingly, NOS levels were signi cantly reduced, and NO showed a non-signi cant downward trend. Thus, taking into consideration the results obtained for both IFN-γ and TNF-α, we are more inclined to believe that NaB downregulates NOS activity by inhibiting the release of TNF-α (Liu, et al., 2019a), thereby reducing NO levels. The peroxynitrite anion (ONOO -) is produced by the rapid binding of NO to oxygen radicals, which may be an important factor in cell damage, energy consumption, and cell death (Ródenas, et al., 1995). We found that levels of ONOOin the bL group were signi cantly downregulated, which contrasts with the elevated levels of NO in this group. Our observations tended to indicate that both IFN-γ and IL-1β and TNF-α are involved in the regulation of the NO-binding peroxide-generating step of ONOO production. And from the analysis just presented, TNF-α and IL-1β are very important in the regulation of ONOO generation (Hou, et al., 2010;Neumann, et al., 2006), particularly at high doses of 7S.
We also found that NaB supplementation signi cantly reduced NOS levels, which may be related to the inhibition of NOS promoter-dependent transcriptional activity by NaB (Sasahara, et al., 2002). Excess ONOO can adversely affect tissues primarily by damaging DNA, inactivating enzymes, and degrading mitochondria (Gerdes, et al., 2020), and thus the downregulation of ONOO in the bH-NaB group may be related to the fact that NaB can enhance the provision of energy to cells (Fang, et al., 2019;Liu, et al., 2017a), thereby preventing the generation of ONOO and thus preventing its negative effects.
As previously mentioned, the strong oxidizing properties of ONOOand its interaction with cytokines such as TNF-α and IL-1β can induce cellular damage and thus disrupt the normal morphology of tissues. To better visualize the effects of NO and ONOO on the intestinal tract of hybrid grouper, we prepared histomorphological sections of the hindgut, which is particularly susceptible to 7S-related disturbance (Periodic acid Schiff and Alcian Blue staining, AB-PAS). Intestinal development in sh can often re ect digestive absorption, and in this regard, plica height is typically used as an indicator of intestinal health.
However, we would argue that plica height alone does not truly re ect the status of intestinal health. Therefore, as an alternative, we used the ratio of intestinal diameter to plica height (Id/Ph), which we believe to be a more reliable indicator of intestinal integrity. The larger this ratio, the greater is the space occupied by folds in the intestines, and thus the greater is the e ciency of absorption (Wang, et al., 2017). On the basis of the ndings of the present study, we established that a high dose of 7S can signi cantly inhibited the development of plicae in the distal intestine of hybrid grouper, thereby reducing the absorptive area of the intestinal fold and thus the e ciency with which feed nutrients are absorbed.
However, supplementing the grouper diet with NaB reversed the detrimental effects of high-dose 7, resulting in a reduction in the Id/Ph ratio, and signi cantly enhancing intestinal development. Similar results have also been obtained for the yellow drum (Nibea albi ora, Richardson) (Wu, et al., 2020a).
The mucus secreted by the mucous cells of sh contains non-speci c immunochemical substances, including lysozyme, transfer factors, chitin, and complementary substances, that are resistant to pathogenic microorganisms (Bosi, et al., 2017). On the basis of AB-PAS staining, mucous cells can be classi ed into four types: I (pure red), II (pure blue), III (purple reddish), and IV (blue purple) . From Figure 2, it can be seen that most of the mucous cells in the distal intestine of hybrid grouper are type II cells. As an acidic mucous cell, the acidic mucus secreted by type II mucous cells plays an important role in regulating the transport of proteins and their residues, lubricating coarse feed, and increasing the viscosity of mucus and immune protection (Gui, et al., 2007). In the present study, we observed that the numbers of type II mucus cells in the intestines of bL and bH-NaB group sh were not signi cantly different from those in the FM group, whereas in contrast, numbers were signi cantly reduced in the bH group, thereby indicating that a high dose of 7S can signi cantly inhibit the formation of type II mucus cells and thus result in the reduced secretion of antimicrobial active substances.
Conversely, supplementing the grouper diet with NaB was found to effectively increase the abundance of type II mucous cells, thereby enhancing the ability of the distal intestine of hybrid groupers to resist pathogenic microorganisms, and thus protecting the distal intestine.
Distal intestinal transduction of the MHC II-PI3K/Akt/mTOR signaling pathway induced by CIITA Subsequent to the ingestion of antigenic proteins in feed, most is converted to amino acids and other small molecules. However, owing to the high molecular weight of 7S, a small fraction is not readily broken down and directly enters the lymphatic system via the intestinal mucosal spaces to induce an immune response (Chen, et al., 2011). When the antigen re-enters the body from the intestines, it will cause intestinal mucosa damage and edema, leading to in ammation. In this regard, although numerous studies have examined the effects of antigenic proteins on the intestines of sh, few have investigated the root cause of the in ammatory effects.
The major histocompatibility complex (MHC) processes foreign antigens, generating antigenic peptide-MHC molecular complexes, which are present in T cells and play an important role in inducing immune responses (Bremberg, 2018). In this process, MHC II and MHC I present exogenous and endogenous antigens, respectively, and in the present study, we focused on the mechanisms associated with the former. In mammals, MHC II encapsulates foreign antigens within vesicular primary endosomes in response to an antigen signal. The principal genes involved in the MHC II antigen presentation process are GILT, AEP, and CTSB (Ganesan, et al., 2015;Maehr, et al., 2005), which function cooperatively to transport the primary endosomes to lysosomes for hydrolysis and antigen degradation. Thereafter, the complexes enter the Golgi apparatus for processing, and are thereby converted to antigenic peptide-MHC II molecular complexes, which activate CD4 T cells and produce corresponding immune responses (Lvaro-Benito, et al., 2018). In this regard, rather than binding directly to the promoter of the MHC II gene, CIITA has been shown to act by controlling MHC II gene transcription via a synergistic interaction with the essential transcription factors RFX5, CREB, and NFY (Gobin, et al., 1998;Wong, et al., 2014). In the present study, we found that the addition of a low dose of 7S promoted the synergistic association between CIITA and CREB1 and NFY to initiate the transcription of MHC II, thus presenting the foreign 7S antigenic protein (Figure 7(a)). In contrast, we detected no signi cant changes in RFX5, and thus MHC I was not upregulated. Studies in patients with type III naked lymphocyte syndrome (BLS) have shown that the RFX complex is required for CIITA-mediated MHC I activity (Gobin, et al., 1998), and it is speculated that this is also the case in hybrid grouper. In response to receiving the antigenic signal from 7S, MHC II forms an intranuclear body regulated by GILT, AEP, and CTSB, and is subsequent to processing within lysosomes and the Golgi system to form an antigenic peptide-MHC II molecular complex, which then transmits the signal to CD4 T cells to activate the immune response. In the distal intestine of hybrid grouper, AEP does not appear to be involved in transduction to nucleosomes, but rather promotes the transport of nucleosomes to lysosomes by upregulating GILT and downregulating CTSB, as has also been observed in mice (Testa, 2010). However, the involvement of AEP appears to be related to the type of antigen detected by an organism, as AEP preferentially deals with viral antigens, as opposed to macromolecular protein antigens (Watts, et al., 2005). CD4 T cells are activated upon receiving signals from the molecular complex, which in turn secrete cytokines with immunomodulatory properties. In combination with the results obtained from our examination of serum-related immunity, these ndings indicate that CD4 T cells and macrophages can enhance intestinal immunity in hybrid groupers by inhibiting the secretion of the pro-in ammatory factors IL-1β and TNF-α and promoting secretion of the anti-in ammatory factor IFN-γ.
The development of intestinal in ammation is generally accompanied by the abnormal expression of constituents of the PI3K/Akt/mTOR pathway (Kim, et al., 2016), the regulation of which by anti-nutritional factor-induced intestinal in ammation is currently incompletely understood. PI3K is a heterodimer composed of the regulatory subunit p85 and the catalytic subunit p110, the activities of which provide a signal for Akt, using PIP3 as a second messenger (Thapa, et al., 2015). mTOR is an atypical serine/threonine protein kinase comprising two complexes, mTOR C1 and mTOR C2. The former consists of three core components, namely, mTOR, Raptor, and mLST8. mTOR is the catalytic subunit of the complex, whereas mLST8 is associated with the catalytic domain of mTOR C1, which stabilizes kinase activation, although is not the catalytic subunit of mTOR C1. mLST8 however, is associated with the catalytic domain of mTOR C1 that stabilizes kinase activation. Rictor does not seem to affect mTOR C1 activity (Lang, et al., 2010). mTOR C2 consists of three core components, namely, mTOR, Rictor, and mLST8, among which mTOR is a component of the catalytic subunit of the complex, as in mTOR C1. mTOR is a rapamycin-insensitive partner that can interact with Protor, although the physiological function of this interaction remains to be determined. mLST8 is a complex catalytic subunit of mTOR C2 (Rosner, Hengstschlager, 2008).
Akt also receives signals from mTOR C2, thereby promoting the expression of the core protein Rheb via the TSC1/TSC2 complex, which regulates mTOR C1 (Hsieh, et al., 2014). On the basis of the low-dose 7S results obtained in the present study, we established that in response to this treatment, Akt is not regulated by mTOR C2 or PI3K, and does not promote Rheb expression via TSC2, thereby disrupting the regulation of mTOR C1. Moreover, in mTOR C1, TEL2 promotes the expression of 4E-BP2, although EIF4E is unaffected, whereas in mTOR C2, TEL2, and Sin1 are likely to be the key components responsible for the upward modulation of RhoA. Furthermore, although the expression of TSC1 is elevated, in combination with Rheb, the regulation of mTOR C1 does not appear to be controlled by Akt. Given the aforementioned observations, we speculate that activation of the PI3K/Akt/mTOR pathway in response to low doses of 7S is super cial and does not have a fundamental effect on key downstream proteins, with CD4 T-cell activation playing a major role.
In response to supplementation with a high dose of 7S (Figure 7(b)), MHC I synthesis is also upregulated via RFX5 activation, and a comparison with the response to low doses indicates that RFX5 is essential for MHC I regulation (Gobin, et al., 1998). Subsequent to the formation of nuclear endosomes, it appears that GILT and AEP, although not GILT and CTSB, are involved in regulation. This observation indicates that the expression of AEP may be dependent not to the type of antigen, but rather the dose of antigen. In this case, following the activation of CD4 T cells by the antigenic peptide-MHC II molecular complex, the secretion of IFN-γ remains at high levels, and there are also signi cant increases in the expression of the pro-in ammatory factors IL-1β and TNF-α, thereby contributing to the development of intestinal in ammation. Interestingly in this regard, we also detected an upregulation of endogenous antigen presentation by MHC I, which we suspect may be associated with the elevated levels of body endogenous antigens induced by high doses of 7S. Likewise, we found that the PI3K/Akt/mTOR pathway was also fully activated in response to stimulation with high doses of 7S. Commencing with PI3K, which activates Akt by upregulating 3-PDK1, we found that IKKα was also upregulated, and although we did not examine the role played by NF-kB in the present study, we speculate that Akt may induce the transfer of NF-κB to the nucleus via IKKα. With respect to mTOR C1, the components depressor, Raptor, and mLST8 are activated, resulting in an mTOR C1-induced upregulation of 4E-BP1 and 4E-BP2, which in turn results in an increase in EIF4E secretion. Moreover, p70 S6K also increased the level of EIF4B via mTOR C1. Upregulation of eIF-4E is often considered a marker of early colon cancer, and it has also been reported that 4E-BP1 and 4E-BP2 limit the anti-in ammatory response of macrophages by inhibiting IL-10 (William, et al., 2018). Thus, high doses of 7S-induced intestinal in ammation in the hybrid grouper may be associated with elevated levels of 4E-BP1 and 4E-BP2 and a suppression of the anti-in ammatory effects of macrophages. In addition, the expression of TSC1 is elevated, thereby in turn promoting the expression of Rheb, which relays signals from Akt to mTOR C1. Furthermore, with respect to mTOR C2, mLST8, depressor, and RICTOR are also activated, thereby elevating the levels of SGK1, which is involved extensively in the regulation of in ammation, and not only induces cardiac in ammation by activating the NLRP3 in ammasome (Gan, et al., 2017) but can also exacerbate in ammation by stimulating NF-kB (Wu, et al., 2020b).
As a common feed additive used to supplement the diets of livestock, poultry, and aquatic animals, NaB can be hydrolyzed in the gastrointestinal tract to form butyric acid, which provides a source of energy to the intestinal epithelial cells, and contributes to the repair of damaged intestinal mucosa and strengthening intestinal immunity. However, the mechanisms whereby NaB in uences intestinal repair have yet to be determined. In the present study, we found that supplementation of feed containing a high dose of 7S with appropriate levels of NaB facilitated the transcription of MHC I and MHC II via the upregulated expression of CREB1 and downregulation of NFY, and that MHC II expression was maintained at a high level (Figure(c)). These observation thus tend to indicate that dietary supplementation with NaB has the effect of enhancing the e ciency of antigenic protein presentation and processing in hybrid groupers. In addition, a concomitant reduction in the expression of MHC I may be indicative of a reduction in the severity of the in ammatory response compared with that seen in groupers receiving a high dose of 7S in the absence of NaB (Wu, et al., 1994). In addition GILT and AEP are inhibited during the development of nucleosomes, which subsequently migrate to the lysosomes and Golgi apparatus. There is also a reduction in expression of the pro-in ammatory factors IL-1β and TNF-α, thereby indicating a weakening of the in ammatory response. Although PI3K expression was also upregulated, the expression of 3-PDK1 and Akt remained at levels similar to those observed in groupers receiving a high dose of 7S. However, we found that the key transcription factor IKKα, which is transduced by Akt to NF-kB, was suppressed. On the basis of these observations, we thus speculate that NaB supplementation could suppress the activation of NF-κB (Si, et al., 2015), although we did not verify this assumption in the present study. With regards to the mTOR C1 complex, we noted an inhibition of mLST8 and Raptor, which in turn downregulated the expression and secretion of 4E-BP1 and EIF4B, and although the expression of p70 S6K was maintained at a high level, the formation of the downstream product EIF4B was inhibited.
Collectively, these ndings indicate that the protective effect of NaB on the hindgut of hybrid grouper is closely associated with small molecule proteins downstream of mTOR C1, such as EIF4E. Although TSC1 showed high expression, that of Rheb was signi cantly reduced, and taken together with the results obtained for groupers receiving feed supplemented with high and low doses of 7S, we speculate that there are probably alternative mechanisms regulating the pathway from Akt to mTOR C1. With respect to mTOR C2, we established that the downregulated expression of mLST8 and Sin1 and upregulation of PRR5 promoted an increase in the expression of RhoA and PKC, and that SGK1 concomitantly remained at high levels. RhoA and PKC are generally considered to function as signal molecules associated with the in ammatory response (Cataisson, et al., 2006;Xie, et al., 2013;Yu, et al., 2019), and are accordingly also commonly used as targets to suppress in ammation (Fard, et al., 2013;Kong, et al., 2013). Thus, NaB does not appear to alleviate intestinal in ammation via mTOR C2, but rather exacerbates the progression of intestinal in ammation.

Conclusion
In the present study, we demonstrated that low and high doses of β-conglycinin (7S) signi cantly enhanced and reduced the weight gain rate, speci c growth rate, and feed utilization of hybrid grouper, respectively. However, we found that supplementation of the grouper diet with sodium butyrate was effective in relieving the growth inhibition caused by high doses of 7S. High-dose 7S was shown to disrupt the development of distal intestinal plica, suppress the density of distal intestinal type II mucus cells, and enhance the synthesis of NO, NOS, and ONOO associated with intestinal injury, thereby exacerbating the process of distal intestinal in ammation. The enhancement of immune responses in the distal intestine of hybrid grouper receiving a low dose of 7S appears to be associated primarily with the activation of CD4 T cells, which promotes the release of the anti-in ammatory factor IFN-γ and reduces that of TNF-α and IL-1β. However, this does not appear to be mediated via by the PI3K/Akt/mTOR pathway. In contrast, this pathway was found to be fully activated upon stimulation with high doses of 7S. Within this pathway, Akt activates mTOR C1 via the promotion of Rheb expression, which in turn enhances the expression of the downstream pro-in ammatory small molecule proteins EIF4E and 4E-BP2, whereas Akt also upregulates SGK1 expression via mTOR C2. These dual effects synergistically contribute to exacerbating in ammation in the distal intestine of hybrid grouper. Furthermore, we established that sodium butyrate relieves intestinal in ammation by inhibiting the expression of the in ammatory-related small molecule proteins 4E-BP2 and EIF4B downstream of mTOR C1. However, in the case of mTOR C2, we found that sodium butyrate does not protect the distal intestine by inhibiting RhoA, PKC, and SGK1, but instead further intensi ed intestinal in ammation by upregulating RhoA and PKC. In combination with the results obtained for growth performance and histopathological analyses, our ndings indicate PI3K/Akt/mTOR C1 and PI3K/Akt/mTOR C2 play major roles in mediating 7Sinduced immune responses in the distal intestine of hybrid grouper, and with respect to the mechanisms underlying the ameliorative effect of sodium butyrate, we suspect that mTOR C1 plays a more important role than mTOR C2. Abbreviations NaB: sodium butyrate; 7S:β-conglycinin; FM group: shmeal group; bL group:low conglycinin group; bH group:high beta conglycinin group; bH-NaB group:high beta conglycinin with sodium butyrate group; kD:Kilo Dalton; ROS:reactive oxygen species; DI:distal intestine; AB-PAS:Alcian Blue-Periodic acid-Shiff; IBW:Initial body weight; FBW:Final body weight; WGR:weight gain rate; SGR:speci c growth rate; SR:survival rate; FCR:feed coe cient ratio; Id/Ph:intestinal diameter/plica height; IFN-γ:interferon γ; IgE:Immunoglobulin E; IL-1β:interleukin-1β; TNF-α:tumor necrosis factor-α; NO:nitric oxide; NOS:nitric oxide synthase; ONOO:peroxynitriteanion; PI3K RS5, phosphatidylinositol 3-kinase regulatory subunit 5; 3-PDK1, 3-phosphoinositide dependent kinase-1; TSC1, tuberous 1; TSC2, tuberous 2; Akt, serine/threonineprotein kinase; IKKα, inhibitor of nuclear factor kappa-B kinase subunit α; Rheb, Ras homolog enriched in brain; Raptor, regulatory associated protein of mTOR; PRAS40, proline-rich Akt1 substrate 1; mTOR, mammalian target of rapamycin; mTOR C1, mammalian target of rapamycin complex 1; mTOR C2, mammalian target of rapamycin complex 2; deptor, DEP domain-containing mTOR-interacting protein; mLST8, target of rapamycin complex subunit lst8; TEL2, telomere length regulation protein; 4EBP1, eukaryotic translation initiation factor 4E binding protein 1; 4EBP2, eukaryotic translation initiation factor 4E binding protein 2; p70 S6K, ribosomal protein S6 kinase β1; EIF4E, translation initiation factor 4E; EIF4B, translation initiation factor 4B; S6, small subunit ribosomal protein S6; Sin1, target of rapamycin complex 2 subunit; RICTOR, rapamycin-insensitive companion of mTOR; PRR5, proline-rich protein 5; RhoA, Ras homolog gene family, member A; PKCα, protein kinase C α; SGK1, serum/glucocorticoidregulated kinase 1; CIITA, major histocompatibility complex class II trans-activator; RFX5, regulatory factor X5; CREB1, cyclic AMP-responsive element-binding protein 1; NFY, nuclear transcription factor Y subunit α; MHC I, major histocompatibility complex class I antigen; MHC II, major histocompatibility complex class II antigen; GILT, gamma-interferon-inducible-lysosomal thiol reductase; AEP, asparaginyl endopeptidase; CTSB, cathepsin B; TCR, T cell receptor;

Declarations
Ethics approval and consent to participate The animal protocol used in the present study was approved by the ethics review board of Guangdong Ocean University, and all procedures were performed in accordance with the standards of the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (NIH Publication No. 8023, revised 1978) and relevant Chinese policies.

Consent for publication
Not applicable.
Availability of data and material All datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.