Ghrelin/GHSR Axis Induced M2 Macrophage and Alleviated Intestinal Barrier Dysfunction in a Sepsis Rat Model by Inactivating E2F1/NF-κB Signaling

Sepsis is an inflammatory reaction disorder state that is induced by infection. The activation and regulation of the immune system play an essential role in the development of sepsis. Our previous studies have shown that ghrelin ameliorates intestinal dysfunction in sepsis. Very little is known about the mechanism of ghrelin and its receptor (GHSR) on the intestinal barrier and the immune function of macrophage regulation. Our research is to investigate the regulatory effect and molecular mechanism of the ghrelin/GHSR axis on intestinal dysfunction and macrophage polarization in septic rats. A rat model of sepsis was established by cecal ligation and puncture (CLP) operation. Then, the sepsis rats were treated with a ghrelin receptor agonist (TZP-101) or ghrelin inhibitor (obestatin). The results suggested that TZP-101 further enhanced ghrelin and GHSR expressions in the colon and spleen of septic rats and obestatin showed the opposite results. Ghrelin/GHSR axis ameliorated colonic structural destruction and intestinal epithelial tight junction injury in septic rats. In addition, the ghrelin/GHSR axis promoted M2-type polarization of macrophages, which was characterized by the decreases of IL-1β, IL-6, and TNF-α, as well as the increase of IL-10. Mechanistically, the ghrelin/GHSR axis promoted E2F2 expression and suppressed the activation of the NF-κB signaling pathway in septic rats. Collectively, targeting ghrelin/GHSR during sepsis may represent a novel therapeutic approach for the treatment of intestinal barrier injury.


Introduction
Sepsis is a clinical syndrome caused by severe infections that always trigger excessive infammatory responses [1].Sepsis, septic shock, and multiple organ dysfunction syndromes (MODS) represent the sequential stages of the disease process [2].Te end stage of MODS is organ failure, which is commonly associated with poor clinical outcomes and a high mortality rate [3,4].During sepsis, the body has an uncontrolled infammatory response to the infection, a large number of infammatory factors are released, multiple infammation-related signaling pathways are activated, and fnally, MODS are observed [5].Tere is an urgent need to fnd new treatment entry points.Excessive infammatory reactions afect the intestine during sepsis, and the intestinal mucosal barrier is damaged [6,7].A large number of bacteria and endotoxins enter the bloodstream, aggravating the infammatory response and promoting the process of subsequent organ damage [8,9].Terefore, the intestinal tract is not only a damaged organ of sepsis but also an important intermediate link in the development of sepsis into multiple organ dysfunctions, so the protection of the intestinal barrier function in sepsis is very important.
Ghrelin is the endogenous ligand of growth hormone secretagogue, which is derived mainly from X/A cells of the gastric mucosa and is an endocrine peptide containing 28 amino acids [10].Ghrelin exerts its biological functions mainly by binding to its receptor GHSR [11].A growing number of studies have shown that ghrelin binds to its receptor and has regulatory efects on growth hormone secretion, feeding, glucose metabolism, cardiovascular system, infammatory response, cell diferentiation, and tumor [12][13][14].It has been reported that patients with sepsis had elevated ghrelin levels, which were inversely related to the length of stay in the ICU and SOFA score [15].Another study found a positive correlation between high serum ghrelin levels and survival in patients with sepsis [16].In animal models of sepsis, ghrelin could reduce infammation, improve gastrointestinal blood perfusion, and reduce gastrointestinal injury in sepsis by regulating autophagy [17].Our previous studies suggested that ghrelin inhibited oxidative stress and intestinal dysfunction to attenuate sepsis by activating SIRT1 and regulating a KLF4/MMP2 regulatory axis [18].Terefore, it can be speculated that ghrelin may be a new treatment for septic gastrointestinal dysfunction.However, the specifc mechanism of its action in sepsis is still unclear.
Te transcription factor E2F1, a member of the E2F family, exerts regulatory control over diverse biological processes and plays pivotal roles in numerous diseases, including cancers, obesity, infammation, and sepsis [19][20][21].Te bioinformatics study demonstrated that E2F1 was a regulator of diferentially expressed genes associated with ferroptosis in sepsis patients [22].E2F1 was implicated in the regulation of the infammatory response to toll-like receptor ligands, including lipopolysaccharide (LPS) [23].Te transcriptional response of the coagulation cascade was diminished in E2F1-defcient mice, suggesting that disseminated intravascular coagulation may be a consequence of uncontrolled sepsis [23].Furthermore, E2F1 could interact with NF-κB forming an E2F1/NF-κB complex.E2F1 inhibited the nuclear translocation of NF-κB p65 by downregulating the phosphorylation level of NF-κB p65, thus deactivating the NF-κB signaling pathway [24].A previous study revealed a positive correlation between the expression levels of ghrelin and E2F1 during cellular proliferation, suggesting that E2F1 may function as a downstream efector of ghrelin [25].Meanwhile, in our previous study, we found that ghrelin increased the expression of E2F1, leading to the inhibition of the NF-κB pathway [26].HDAC5 inhibitor LMK-235 suppressed infammation in sepsis via the ghrelin/E2F1/NF-κB axis [26].
In the present study, we investigated the efects of ghrelin on colonic infammation, intestinal barrier function, and immune function in septic rats using ghrelin receptor agonist (TZP-101) and ghrelin inhibitor (obestatin).Furthermore, we explored the regulatory efect of ghrelin on the E2F1/NF-κB signaling pathway in the colon of septic rats.

Methods and Materials
2.1.Animals.Male Sprague-Dawley (SD) rats were purchased from Chengdu Dossy Experimental Animals Co., Ltd.(Chengdu, Sichuan).Te feeding environment was 25 ± 1 °C, relative humidity was 50%-60%, and light/darkness for 12 h circulation.Rats were allowed to eat and drink freely.Animals and experimental protocol were conducted according to the guidelines and ethical standards of the Animal Care and Use Ethics Committees of Lanzhou University (LDYYLL2021-87).

Sepsis Models.
A rat model of sepsis was established by cecal ligation and puncture (CLP) according to a previously described protocol [27,28].Rats were fasted for 12 h before the experiment and had free access to water.Anesthesia was induced by intraperitoneal injection of 1% sodium pentobarbital 50 mg/kg.Anesthesia depth was determined by the pinch test.Laparotomy was used to expose the cecum postgeneral anesthesia.Te cecum was gently picked out and ligated with a 4-gauge thread at the 2/3 cecal end to avoid ligation of blood vessels.Te cecum was gently perforated once with an 18 G trocar, with two perforations, and little feces was gently extruded to avoid injury to the mesenteric vessels.Te cecum was then put back in the abdominal cavity, and the abdominal wall incision was sutured layer by layer to sterilize the incision.

Experimental Design.
Tirty-six rats were randomly divided into the normal group, sham operation group, CLP group, ghrelin treatment group, ghrelin + ghrelin receptor agonist (TZP-101) group, and ghrelin + ghrelin inhibitor (obestatin) group, with 6 rats in each group.Rats in the normal group, sham operation group, and CLP group were intraperitoneally injected with 1 ml of normal saline at 2 h after the operation.Rats in the ghrelin group were intraperitoneally injected with ghrelin 20 ng/kg 2 h after the operation, and colon tissues and spleen tissues were collected 12 h after drug intervention.Rats in the ghrelin + TZP-101 group were injected intraperitoneally with ghrelin 20 ng/kg and intravenously with TZP-101 1 mg/ kg 2 h postoperation, and colonic tissues and spleen tissues were taken 12 h after drug intervention.Ghrelin + obestatin group rats were injected with ghrelin 20 ng/kg intraperitoneally and obestatin 100 nmol/kg intraperitoneally at 2 h postoperation, and colonic tissues and spleen tissues were taken after 12 h of drug intervention.

Hematoxylin and Eosin (H&E) Stain.
Te colonic tissues of rats were collected and fxed in 4% paraformaldehyde overnight, processed, and embedded in parafn.Te tissue sections were stained with hematoxylin and eosin (H&E) to observe the degree of the lesion and infammatory cell infltration under a 200x and 400x magnifcation optical microscope (Olympus BH2, Tokyo, Japan).

Periodic Acid-Schif (PAS) Stain Reaction Stain.
Following the manufacturer's instructions, PAS staining was performed with the PAS Staining Kit (Muto Pure Chemicals, Tokyo, Japan).Colonic tissues were fxed with 10% (w/v) 2 Canadian Journal of Gastroenterology and Hepatology formaldehyde and treated with 1% (w/v) periodic acid for 10 min at room temperature.After the sections were washed three times with distilled water, they were treated with Schif's reagent for 30 min at 37 °C.Ten, sections were rinsed using distilled water for 10 min, and nuclei were stained with hematoxylin for 1.5 min.Te sections were rinsed using running water and sealed.Te sections were examined by light microscopy.

Enzyme-Linked Immunosorbent Assay (ELISA).
Te levels of LPS from rat serum samples and colonic tissues were examined with an ELISA kit (Zhuocai Biological Technology, China) based on the manufacturer's instructions.Te tissue was homogenized using a tissue grinder, the homogenate was centrifuged at 5000 × g for 10 min, and the supernatant was taken for measurement.Te samples were plated for 30 min at room temperature onto a microplate precoated with the antibody specifc for LPS.After the immune complex was treated, the absorbance of wells was measured with a microplate reader (SpectraMax Plus 384, USA) at 450 nm wavelength to calculate the sample concentration according to the manufacturer's instructions.

RT-qPCR Assay.
Te mRNA expression of TNF-α, IL-1β, IL-6, E2F1, IκB, and p65 was evaluated by using RT-qPCR.For gene analysis, equal amounts of cDNA were added to a reaction mixture containing gene-specifc forward and reverse primers deoxynucleotide Taq DNA polymerase and SYBR (Bio-Rad, Hercules, CA) in a reaction mixture.Quantifcation of cDNA was based on monitoring increased SYBR fuorescence during exponential phase amplifcation in an RT-qPCR machine (Bio-Rad, Hercules, CA), and the determination of the PCR cycle number at which the amplifed product exceeded a defned threshold.
2.12.Statistical Analysis.Te results of the experiments were statistically analyzed using SPSS analytical statistical software.Te experimental results were expressed using the mean ± standard deviation (SD).One-way analysis of variance (ANOVA) was used for statistical analysis.A diference of p < 0.05 was defned as signifcant.

Ghrelin/GHSR Axis Ameliorated Colonic Structural Destruction in Septic
Rats.Te growth hormone secretagogue receptor (GHSR) was characterized as a member of the Gprotein-coupled receptor family and as a newly founded receptor of ghrelin [29].Our results found that the expression of ghrelin and GHSR was decreased in the colon and spleen of septic rats which was induced by ghrelin treatment (Figures 1(a)-1(d)).Meanwhile, TZP-101 further enhanced ghrelin and GHSR expressions in the colon and spleen of septic rats and obestatin showed the opposite results (Figures 1(a)-1(d)).Tese results suggest that the ghrelin/GHSR axis may regulate the gastrointestinal function and immune function in septic rats.
A histological examination was performed after staining with H&E or PAS.CLP treatment caused extensive exfoliation of crypt epithelial cells, crypt destruction, and loss of glandular structure (Figure 1(e)).Te ghrelin treatment group ameliorated the disruption of colonic structure caused by CLP treatment (Figure 1(e)).In addition, compared with the ghrelin-treated group, ghrelin and TZP-101 cotreatment further reduced the disruption of colonic structure.
Canadian Journal of Gastroenterology and Hepatology However, obestatin hindered the ameliorative efect of ghrelin on the colon of septic rats (Figure 1(e)).As shown in Figures 1(b) and 1(c), the colons of CLP mice had fewer goblet cells compared with the control and sham groups.Ghrelin treatment enhanced the number of goblet cells compared with the CLP group (Figures 1(f ) and 1(g)).Furthermore, ghrelin and TZP-101 cotreatment further increased the number of goblet cells compared with the ghrelin-treated group.Te cotreatment of ghrelin and obestatin showed the opposite results (Figures 1(f ) and 1(g)).Te higher number of goblet cells in the ghrelin and ghrelin + TZP-101 group may have protected sepsis rats from CLP-induced injury.

Ghrelin/GHSR Axis Attenuated Intestinal Epithelial Tight
Junction Injury in Septic Rats.Te intestinal permeability of septic rats was increased, and the content of FTC-glucan in serum was signifcantly higher than that in control and sham groups (Figure 2(a)).Ghrelin treatment decreased the content of FTC-glucan in the serum of septic rats, which was reversed by obestatin (Figure 2(a)).LPS, a component of the intestine that triggers infammation, plays a role in mucosal barrier function [30,31].Ten, the levels of serum LPS (Figure 2

Ghrelin/GHSR Axis Promoted M2-Type Polarization of
Macrophages.We then examined the efect of the ghrelin/ GHSR axis on macrophage polarization in the colon and spleen of septic rats.CD86 and CD206 are specifc markers of M1 macrophages and M2 macrophages, respectively.IHC Canadian Journal of Gastroenterology and Hepatology stain showed that the expression of CD86 was signifcantly increased and that of CD206 was signifcantly decreased in the colon and spleen of septic rats (Figures 4(a)-4(h)).In addition, CD86 expression was decreased and CD206 expression was increased in the colon and spleen of ghrelintreated septic rats (Figures 4(a)-4(h)).TZP-101 reduced and obestatin enhanced CD86 expression in the colon and spleen of septic rats compared with the ghrelin alone treatment group (Figures 4(a)-4(h)).As expected, TZP-101 promoted and obestatin inhibited CD206 expression in the colon and spleen of septic rats compared with the ghrelin alone treatment group (Figures 4(a)-4(h)).Tese results suggest that the ghrelin/GHSR axis promoted M2-type polarization of macrophages in the colon and spleen of septic rats.Te ghrelin/GHSR axis activated E2F1 expression and suppressed the activation of the NF-κB signaling pathway in septic rats.
Finally, we explored the molecular mechanisms by which ghrelin ameliorates gastrointestinal dysfunction in sepsis.Te mRNA level of E2F1 was decreased and the mRNA levels of IκB and NF-κB p65 were increased in the colon of septic rats (Figures 5(a)-5(c)).Furthermore, ghrelin promoted the E2F1 mRNA level and inhibited the mRNA levels of IκB and p65 compared with the CLP group (Figures 5(a

Discussion
Severe sepsis of abdominal origin leads to the impairment of intestinal barrier integrity, which is mainly manifested as enhanced intestinal mucosal permeability, intestinal mucosal perfusion disorder, tissue edema, and bacterial translocation [32,33].Te destruction of intestinal epithelial cell function in patients with severe abdominal sepsis is a key factor causing septic shock and multiple organ dysfunction syndrome [34].Te structure of the intestinal epithelium with its tight junction disposal allows only the passage of very tiny molecules, preventing bacterial or macromolecular (e.g., LPS) transport [35].Under normal circumstances, the intestinal tract absorbs nutrients while maintaining bacteria within the intestinal lumen.In sepsis, the changes in the intestinal permeability to macromolecular follow the same time course as bacterial overgrowth and increased toxin production, and the intestine may allow increased bacterial infltration into mesenteric lymph nodes (MLNs) and other extraintestinal sites (e.g., spleen, lung, liver, and blood) [7,36,37].Our results showed that crypt epithelial cells were shed and crypt architecture was disrupted in the intestinal tissues of septic rats, which were reversed by treatment with ghrelin and ghrelin receptor agonist TZP-101.Furthermore, paracellular bacterial transport may also be facilitated by 6 Canadian Journal of Gastroenterology and Hepatology changes in intestinal epithelial cell structure, particularly involving tight junctions [38].Te tight junction is mainly composed of various tight junction proteins, such as occludin, claudin-5, and ZO-1 [39,40].Te study suggested that serum ZO-1 could serve as a robust biomarker for assessing gut barrier dysfunction in sepsis [41].In a study investigating the impact of berberine pretreatment on sepsis, occludin, ZO-1, and claudin-4 were found to exhibit decreased expression levels following CLP in rats [42].Meanwhile, irisin upregulated occludin and ZO-1 in lung tissues of sepsis-induced acute lung injury rats [43].To the best of our knowledge, this study represented a pioneering demonstration that ghrelin and TZP-101 could enhance claudin-5 and ZO-1 levels in the intestinal tissue of septic rats.Tus, our results suggested that the ghrelin/GHSR axis attenuated intestinal barrier permeability and intestinal epithelial tight junction injury in septic rats, which was consistent with our previous fndings [18].Te dysregulation of infammation serves as the fundamental mechanism underlying sepsis pathogenesis and persists throughout sepsis [44].Te anti-infammatory properties of ghrelin have been demonstrated in several in vivo studies.In septic mice, ghrelin decreased cerebral edema and improved the blood-brain barrier integrity by decreasing infammation [45].Another study reported that ghrelin improved ventricular peak systolic pressure and cardiac contractility of CLP rats by reducing proinfammatory cytokines [46,47].In CLP-induced septic mice, 8 Canadian Journal of Gastroenterology and Hepatology ghrelin administration improved infammatory cytokine levels, kidney function, and arterial blood pressure [48].On the other hand, TNF-α and IL-6 levels in rats were decreased following CLP surgery by administration of ghrelin [49].After 12-24 h after CLP, ghrelin signifcantly improved survival in mice and reduced clinical parameters and histopathological scores of sepsis, thus demonstrating its efects on late mediators of infammation [50].Similarly, we found that in CLP rats, ghrelin administration produced signifcant inhibition of the release of TNF-α, IL-6, and IL-1β, as well as increased levels of IL-10 in colon tissues.Tese fndings align with our previous study which found that ghrelin ameliorated CLP-induced sepsis in mice by reducing infammation and weight loss [51].In conclusion, our study further clarifed the inhibitory efect of ghrelin on intestinal infammation in septic rats.
Te main contributors to infammatory cytokine production in sepsis are reported to be macrophages [52].Macrophages possess the ability to perform chemotaxis, antigen presentation, phagocytosis, and pathogenic bacteria eradication, while also serving the crucial role of regulating the infammatory response to uphold homeostasis, thereby establishing their signifcance as a pivotal defense mechanism within the human body [53,54].Macrophages are believed to be extremely plastic [55].In the early stages of sepsis, which is characterized by a systemic infammatory response, infection triggers the diferentiation of macrophages to M1-type macrophages, inducing the secretion of a large number of infammatory factors that participate in the pathogenic bacterial killing and clearance process [56].In the later stage of sepsis characterized by immunosuppression, macrophages diferentiate into M2-type macrophages, which highly express anti-infammatory cytokines and participate in tissue repair to control excessive infammatory response [57].Tus, targeted regulation of macrophage polarization may ofer new treatment modalities for sepsis.Zhang et al. [58] recently found that monocyte chemotactic protein (MCP)-induced protein 1 (MCPIP1) regulated M2-type macrophage polarization through inhibition of the JNK/c-Myc signaling pathway, thus reducing sepsis-induced acute lung injury and mortality.Chronic Schistosoma japonica (SJ) infection had a protective efect in septic mice by enhancing M2-type macrophage polarization and inhibiting M1-like macrophage polarization [59].In addition, T-cell immunoglobulin mucin 3 (Tim-3) inhibited M1-type macrophage polarization, thereby reducing proinfammatory activity in the early stage of LPS-induced sepsis [60].However, in the later stage of sepsis, a decrease in Tim-3 expression inhibited M2-like macrophage polarization and promoted M1-type macrophage polarization, respectively [60].In our study, the ghrelin/GHSR axis inhibited M2-type polarization of macrophages and promoted M2-type polarization of macrophages in the colon and spleen of septic rats.Ultimately, these reduced anti-infammatory factors release and avert infammatory insults.
Furthermore, our study creatively identifed the potential of E2F1/NF-κB signaling to inhibit sepsis-induced intestinal dysfunction in vivo.Te correlation between p65 and E2F1 has been previously confrmed in human fbroblasts, where this physical interaction hinders the expression of E2F-responsive genes [61].Similarly, E2F1 has been documented to hinder the antiapoptotic NF-κB signaling pathway by diminishing the levels of TNF receptorassociated factor 2, a key activator of NF-κB.Tis inhibition occurs either through competitive binding with p50 for RelA/ p65 in murine fbroblasts or by suppressing IκB phosphorylation [62][63][64].Likewise, the interplay between E2F1 and NF-κB was reported to regulate infammation in human cardiac cells [65].E2F1 inhibited the binding of NF-κB p65 to the ICAM-1 promoter and eliminated the antitumor immune efect of ICAM-1 against prostate cancer cells [66].It was reported that NF-κB is a pleiotropic transcription factor implicated in the regulation of sepsis and septic shock [67].Inhibition of miR-155 alleviated infammation and intestinal barrier dysfunction by suppressing NF-κB signaling in mice with sepsis [68].miR-199a-5p exacerbated intestinal barrier dysfunction by promoting the activation of the NF-κB pathway [69].In addition, the study found that silencing of miR-31 protected against intestinal barrier dysfunction by inhibiting the NF-κB/HIF-1α pathway and targeting HMOX1 during sepsis [70].Furthermore, somatostatin (SST) repaired sepsisinduced intestinal barrier dysfunction through suppression of NF-κB signaling [71].Notably, our data suggested that the ghrelin/GHSR axis promoted E2F1 expression and suppressed the NF-κB signaling pathway in septic rats.To the best of our knowledge, the present study is the frst to report the inhibitory efect of the ghrelin/GHSR axis on the E2F1/NF-κB signaling pathway in a rat model of sepsis, which implies that the protective efect of ghrelin/GHSR axis on sepsis-induced intestinal barrier dysfunction was mediated by eliminating the activity of E2F1/NF-κB signaling pathway.
Taken together, our results reveal a modulatory efect of the ghrelin/GHSR axis on intestinal barrier function and immune function in sepsis.Te ghrelin/GHSR axis increased intestinal barrier function by eliminating tight junction damage.Furthermore, the ghrelin/GHSR axis suppressed the CLP-induced infammatory response by promoting M2type polarization of macrophages.Te mechanisms underlying the efects of the ghrelin/GHSR axis may be related to the regulation of the E2F1/NF-κB signaling pathway.Tus, the ghrelin/GHSR axis can be a potential therapeutic target for treating human sepsis.
)-5(c)).TZP-101 treatment increased E2F1 mRNA level and inhibited IκB mRNA level compared with the ghrelin alone treatment group.Obestatin treatment showed the opposite results (Figures 5(a)-5(c)).Detection of protein levels was performed with Western blot analysis.As shown in Figures 5(d)-5(f ), the results showed that the expression of E2F1 was downregulated and the phosphorylation of IκB and p65 was upregulated in the colon of septic rats.Ghrelin activated E2F1 expression and suppressed the phosphorylation of IκB and p65 (Figures 5(d)-5(f )).In addition, TZP-101 treatment increased E2F1 expression and inhibited the expression of IκB in the cytoplasm and p65 in the nucleus compared with the ghrelin alone treatment group (Figures 5(d)-5(f )).On the contrary, obestatin resulted in decreased E2F1 expression, leading to increased phosphorylation of IκB and p65 compared with the ghrelin alone treatment group (Figures 5(d)-5(f )).Finally, IF staining was performed to examine the nuclear localization of p65.Compared with the CLP group, nuclear localization of p65 was downregulated signifcantly by ghrelin, and TZP-101 inhibited and obestatin promoted this efect (Figures 5(g)-5(h)).