Introduction
Hepatic ischemia-reperfusion injury (HIRI) is a major form of liver damage that can occur during surgeries, such as partial hepatectomy and liver transplantation,1 and with conditions such as hemorrhagic shock and severe infection.2 It has two stages: initial cellular damage due to hypoxia and the subsequent restoration of oxygen delivery, and subsequent toxic effects of oxygen reperfusion on ischemic tissue. These effects can further exacerbate liver dysfunction and damage, leading to processes such as inflammatory cell infiltration, the production and release of cytokines and chemokines, oxygen-free radical production and destruction, and mitochondrial Ca2+ overload.3–7 Despite extensive research on HIRI, effective drugs for clinical use are lacking, and the identification of viable drug targets is critical for the development of clinical therapies. G protein–coupled receptors (GPCRs) form the largest class of cell surface receptors in humans and a major drug target category. Our research focuses on the identification of GPCRs associated with HIRI, with the aim of discovering new therapeutic targets to guide effective drug development and provide novel approaches to the clinical treatment of HIRI.
We are particularly interested in GPCRs that have been shown to regulate inflammation and injury repair, such as calcitonin gene–related peptide (CGRP) receptors. Receptor activity-modifying protein 1 (RAMP1) and calcitonin-like receptor (CLR) are the two main components of these receptors, involved in processes such as vasodilation, pain stimulation, and inflammation.8 RAMP1 has been studied extensively due to its ability to reduce inflammation,9,10 and promote injury repair.11,12 As the alteration of its expression can modulate the sensitivity of CGRP, and as it binds to and regulates CLR expression, the targeting of RAMP1 may be a promising approach to the alteration of CGRP activity.13 RAMP1-deficient mice exhibit impaired organ mass recovery and hepatocyte proliferation, which stimulates the expression and activity of yes-associated protein (YAP)/TAZ after 70% hepatectomy or repeated intraperitoneal injection of carbon tetrachloride.14 Additionally, RAMP1 is involved in the regulation of apoptosis, and the in-vitro and in-vivo inhibition of the CGRP/CRLR + RAMP1 signaling pathway can induce apoptosis in EVI1-high AML cells by disrupting extracellular signal–regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) signaling.15 The ERK/MAPK pathway is known to be involved in the regulation of various cellular processes, including cell proliferation, differentiation, and apoptosis.16 The excessive activation and increased expression of ERK1/2 may promote apoptosis and worsen IRI.17–19 The investigation of whether the expression of RAMP1 is altered during HIRI and whether RAMP1 affects apoptosis through the ERK/MAPK pathway is crucial. Whether RAMP1 deficiency affects YAP phosphorylation in this context is also worth exploring. Thus, we investigated the role of ERK1/2 in HIRI.
This study revealed that RAMP1 expression is significantly increased in the livers of mice treated with liver I/R, suggesting that RAMP1 plays a role in this process. Our in-vivo and in-vitro experiments showed that RAMP1 alleviates liver injury by reducing hepatocyte apoptosis and the inflammatory response via the ERK/MAPK pathway. Moreover, we found that RAMP1 decreases YAP phosphorylation, thereby promoting hepatocyte activity. Our findings suggest that RAMP1 serves as a pivotal regulator in HIRI and is a promising target for clinical intervention.
Methods
Animals
Male RAMP1 knockout (RAMP1-KO) mice aged 6–8 weeks were obtained from Jiangsu Gempharmatech Biological Science and Technology Ltd (Nanjing, Jiangsu, China) and bred at the Guangzhou Ruiye Animal Model Center. CRISPR-Cas9 technology was used to edit RAMP1 in the KO mice. All mice were maintained on a 12/12-hour light/dark cycle and provided with food and water.
Hepatic ischemia-reperfusion injury mouse models
A non-lethal segmental (70%) hepatic warm ischemia-reperfusion model was established in both WT and RAMP1-KO mice.20 Microvascular clamps were used to block the first porta hepatis for 90 min, followed by reperfusion. Mice were sacrificed at 0, 6, and 24 h after reperfusion, and liver and blood samples were collected. The sham group underwent identical procedures but without the hepatic portal vein being blocked.
Immunohistological and the terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL) staining
Immunohistological staining of liver samples was performed according to the manufacturer’s protocol. Briefly, following a sequence of fresh xylene, 100 % ethanol, 100 % ethanol, 95 % ethanol, and 75 % ethanol passing through the cylinder, along with EDTA antigen repair, the slides were then incubated with RAMP1 antigen antibodies (ab203282, abcam) and Ki67 antibodies (ab15580, abcam). Subsequently, the sections were stained with Dako secondary antibody (DAKO immunohistochemical kit REAL EnVision) and counterstained with hematoxylin. Hematoxylin and eosin (H&E) staining were used to observe pathological changes in the liver ischemia-reperfusion area of the mice. The TUNEL assay was performed using an in situ cell death detection kit (11684817910, Roche).21 Nuclei were stained with DAPI (62248, ThermoFisher Scientific). Nuclei with clear red staining indicated TUNEL-positive apoptotic cells. The mean ± standard deviation (SD) of positive cells per 1,000 samples was calculated, followed by counting the labeled cells in 10 fields of view at 200× magnification.
Liver function assay and histologic examination
Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured as indicators of liver injury using an automatic analyzer (Antech Diagnostics, Los Angeles, California, USA). Paraffin-embedded formalin-fixed liver tissue was cut into 4-µm thick sections. The sections were stained with (H&E),22 and inflammation and tissue damage were blindly analyzed using Suzuki’s standard.23
Primary hepatocyte isolation
As described previously,21,22,24 primary hepatocytes were isolated from mice aged 6–8 weeks. Briefly, after anaesthesia, the mice were infused with a buffer solution (lacking Ca2+ and Mg2+) through the portal vein. Liver perfusion was performed using 0.05% type IV collagenase (C5138, Sigma), followed by resection and filtration through a 0.22-µm cell filter (SLGP033RB, Millipore). Hepatocytes were isolated and collected in DMEM (C11995, GIBCO).
Cell culture and hepatocyte hypoxia/reoxygenation (H/R) model
Primary hepatocytes or L02 cells were cultured in DMEM/F12 containing 10% fetal bovine serum (FBS) in a humidified atmosphere of 5% CO2 at 37°C. The I/R model was simulated in vitro and the cells were treated with H/R. Primary hepatocytes initially placed in glucose-free DMEM (C11995, GIBCO) were cultured under hypoxic conditions (1% oxygen) for 4 h, followed by exposure to normoxic conditions.21
Western blot analysis
Total protein was extracted from the liver tissue and primary hepatocytes following the manufacturer’s protocol. The samples were then separated on 12% sodium dodecyl sulfate-polyacrylamide gels (Bio-Rad, Hercules, CA, USA) and transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% skim milk powder at 25°C for 1 h and incubated overnight at 4°C with the following primary antibodies targeting the following proteins: RAMP1(ab156575, abcam), Bcl-2 (Cat. #3498S, Cell Signaling Technology [CST]), Bax (Cat. #2772S, CST), Caspase-3 (Cat. #9662S, CST), ERK1/2 (Cat. #9102S, CST), p-ERK1/2 (Cat. #9101S, CST), JNK (Cat. #9252S, CST), p-JNK (Cat. #4668S, CST), P38 (Cat. #8690S, CST), p-P38 (Cat. #4511S, CST), YAP (Cat. #14074S, CST), p-YAP (Cat. #4911S, CST), β-actin (Cat. #4967S, CST).
Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was isolated using TRIzol (15596026, ThermoFisher), while cDNA synthesis was carried out using the Hiscript III RT Supermix for QPCR (+gDNA Wiper) kit, following the manufacturer’s instructions. Subsequently, the cDNA was quantified using a LightCycler 480 high-throughput real-time fluorescent quantitative PCR instrument. The mRNA expression level was normalized to 18s rRNA level. Primers for qRT-PCR were synthesized by Tsingke Company in Wuhan.
Cell Counting Kit-8 assay (CCK-8)
Adherent primary hepatocytes were plated in 96-well plates, and each group was spread with 3–5 holes. Each hole was filled with 200 µL medium or configured drugs. During the detection, the culture medium was replaced, 10 µL CCK-8 (KGA317-2, KeyGEN) and 100µL culture medium (C11875500BT, GIBCO) were added to each well, and incubated at 37°C for 2 h. Finally, the absorbance at 450 nm was measured using a microplate reader.
Drug
YAP phosphorylation inhibitor: Truli25 (E1061, Selleck, 0.2 nm) and PY-6026 (HY-141644, MCE; 1.6 µm; 10 mg/kg for mice in vivo, i.p.); ERK phosphorylation inhibitor: Temuterkib27 (HY-101494, MCE; 5 nm) and SCH77298428 (HY-50846, MCE; 300 nm, 5 mg/kg for mice in vivo, i.p.29); ERK agonist: C16-PAF(HY-108635, MCE, 1 µm30,31); STAT3 inhibitor : Sttatic32 (HY-13818, MCE; IC50:10 µm); p-AKT inhibitor: MK220633 (HY-10358, MCE; 65 nm); Caspase-3 inhibitor: Z-VAD (HY-16658B, MCE; 10 µm34; 10 mg/kg for mice in vivo, i.p.35); CGRP agonist: Calcitonin Gene Related Peptide (CGRP) II, rat TFA (HY-P1913A , MCE; 83 µm36); Verteporfin (HY-B0146, MCE; 5 µm37).
Caspase-3 activity assay
Caspase-3 activity assays were conducted using the Caspase-3 Activity Assay Kit (C1115, Beyotime), following previously established protocols.38,39 Briefly, liver tissue treated with pyrrolidine lysate was lysed using lysis buffer. The supernatants obtained from the homogenates were collected by centrifugation at 16,000 g for 15 min, and the protein concentration was quantified using the Bradford Protein Assay kit (P0006, Beyotime). Subsequently, the lysates were incubated with Ac-DEVD-pNA (2 mmol/L) at 37°C for 2 h. After incubation, the absorbance was measured at 405 nm using a microplate reader (BioTek).
Caspase-3 Activity and Apoptosis Detection Kit for Live Cell (C1077M, Beyotime)
Following H/R, the cell culture medium was aspirated into a suitable centrifuge tube and the cells were rinsed once with PBS. An appropriate volume of trypsin cell digestion solution was added for cell detachment. The cells were collected, transferred to a centrifuge tube, and centrifuged at 2,000 rpm for 5 min, The supernatant was discarded, and the cells were gently suspended in PBS. The resuspended cells were centrifuged at 2,000 rpm for 5 min, and the supernatant was discarded. In total, 194µL Annexin V-mCherry Binding Buffer was added to gently resuspend the cells. Furthermore, 5 µL Annexin V-mCherry and 1µL GreenNuc™ Caspase-3 Substrate (1 mM) were added and mixed gently. The mixture was incubated at room temperature (20–25°C) for 20–30 min in the dark. GreenNuc™-DNA exhibited green fluorescence (excitation/emission=500/530 nm), whereas Annexin V-mCherry displayed red fluorescence (excitation/emission=587/610 nm). Cells were collected and gently suspended in 100 µL Annexin V-mCherry Binding Buffer. After smearing, the cells were observed using an Elyra 7 Lattice SIM (Zeiss, Germany). We followed identical procedures for flow cytometry experiments, employing Gallios (Beckman Coulter) flow cytometers for the analyses, and the data were analyzed using CytExpert software packages.
Flow cytometric analysis of apoptotic cell
Cells were collected as described previously. The supernatant was discarded and 2 µL Propidium iodide (PI) and 2 µL Annexin V (KGA107, KeyGEN) were added and mixed gently. Cells were suspended with 250 µL Binding Buffer and incubated at room temperature (20–25°C) for 20–30 min in the dark. Flow cytometric analyses were performed using a Gallios flow cytometer (Beckman Coulter), and the data were analyzed using the CytExpert software package.
Immunofluorescence staining
To analyze immune cell accumulation in the liver, immunofluorescence staining of monocytes was performed using primary antibodies against mouse CD68 (ab125212; abcam) and RAMP1 (ab203282; abcam). Goat anti-rabbit and anti-mouse IgG (Invitrogen) were used as the secondary antibodies. A 488 nm wavelength laser was employed to excite the fluorescein tag (green emission for imaging), and a 555 nm wavelength laser was used for the fluorescein tag (red emission for imaging) (G1236-100T, Servicebio). DAPI was excited using UV light (blue emission for imaging). The images were obtained using three emerging channels. GreenNuc™-DNA and Annexin V-mCherry (C1077M, Beyotime) were used to monitor Caspase-3 activity and apoptosis in living cells, and images were generated from both channels. The co-localization of RAMP1 and macrophages was detected using confocal microscopy (LSM980, Zeiss, Germany).
Transmission electron microscopy
The anaesthetized mice were perfused with 0.9% saline, and 1 mm3 liver tissue blocks were obtained. Next, the fresh tissue blocks were immersed in fixative for transmission electron microscopy (Servicebio) at 4°C for 4 h. They were then treated with 1% OsO4 in 0.1 mol/L PBS for 2 h at room temperature, followed by a dehydration process with gradient alcohol. Subsequently, the sections were embedded and underwent baking in an oven at 60°C for 48 h and were cut into ultrathin sections (60 nm) using an ultramicrotome. Finally, the ultrastructure of the tight junctions of the liver was observed using a transmission electron microscope (HITACHI, HT7700) as described previously.40
Statistical analyses
Data were expressed as the means ± SDs, including IHC staining and western blot results. Statistical differences between the two groups were analyzed using a two-tailed unpaired Student’s t-test. p<0.05 was considered statistically significant by GraphPad Prism 8.0.1 (GraphPad Software, USA).
Results
RAMP1 upregulation was related closely to HIRI
To examine the changes in RAMP1 in HIRI, we created an in-vivo model of HIRI in mice and an in-vitro model of hypoxia/reoxygenation (H/R) treatment of primary hepatocytes isolated from mice. RAMP1 expression was upregulated at the protein and mRNA levels in the HIRI model and peaked at 6 h after I/R treatment (Fig. 1A–C). Primary hepatocytes from wild-type (WT) mice treated with H/R also showed increases in RAMP1 protein levels compared with control hepatocytes (Fig. 1D, E). Immunohistochemical analysis confirmed that RAMP1 expression peaked at 6 h after I/R treatment (Fig. 1F, G).
RAMP1 ameliorates liver damage induced by HIRI
To investigate the role of RAMP1 in HIRI in vivo, we generated RAMP1- knockout (KO) mice. The mice exhibited RAMP1 deficiency in the liver (Fig. 2A) and hepatocytes (Fig. 2B), and showed no signs of liver damage under sham conditions (Fig. 2C). Following the HIR operation, RAMP1-KO and WT mice had increased serum ALT and AST levels reflecting liver damage, which were higher in the former group (Fig. 2C, D). Histological analysis of liver tissue obtained immediately after reperfusion showed normal hepatocyte morphology and lobular structure in the sham group, and mild to moderate hepatocyte swelling and mild hepatic sinus dilation in the WT and RAMP1-KO groups. At 6 h after reperfusion, severe hepatocyte swelling, inflammatory cell infiltration, and patchy necrosis were observed in the RAMP1-KO and WT groups, and were more serious and extensive in the former (Fig. 2E, F). Consistent with these histological assessments, the necrotic area and Suzuki score were significantly elevated in the RAMP1-KO group compared with those in the WT group (Fig. 2G). Levels of the inflammatory factors IL-6, IL-1β, and TNF-α, detected by quantitative RT-PCR (Supplementary Table 1), showed similar changes over time in both groups (Supplementary Fig. 1A, C, D). However, the level of the protective inflammatory factor IL-10 was significantly decreased in the RAMP1-KO group at 6 h after reperfusion (Supplementary Fig. 1B). These findings suggest that RAMP1 plays a protective role against HIRI in mice.
RAMP1 inhibited hepatocyte apoptosis and promoted liver proliferation in HIRI
As apoptosis is the primary manifestation of HIRI, we determined the apoptotic rate in liver tissues using TUNEL staining. We found that RAMP1 deficiency worsened apoptosis, especially at 6 h after I/R (Fig. 3A, B). Under sham conditions, TUNEL signals were undetectable in WT and RAMP1-KO mouse livers. Relative to those of WT mice, the livers of RAMP1-KO mice showed reduced expression of Bcl-2 (a pro-survival gene) and increased expression of Bax (a pro-apoptotic gene) (Fig. 3C, Supplementary Fig. 2A). Western blot analysis revealed that HIRI induced greater expression of cleaved Caspase-3 in the RAMP1-KO group than in the WT group (Fig. 3C, Supplementary Fig. 2B). The same phenomenon was observed in primary hepatocytes (Fig. 3D, Supplementary Fig. 2C, D). Transmission electron microscopy also showed distinct indications of apoptosis (Supplementary Fig. 3A, B).
Conversely, significantly fewer Ki67-positive cells were observed in the RAMP1-KO group than in the WT group (Fig. 3E, F). We examined alterations in Caspase-3 activity during HIRI and used Annexin V staining to detect apoptosis (Fig. 4A, B). The red and green fluorescence of hepatocytes increased notably during hepatic I/R and weakened upon treatment with Z-VAD (a Caspase-3 inhibitor). Flow cytometry demonstrated significant increases in apoptosis and Caspase-3 activity during H/R and a decrease in the proportion of apoptotic cells upon Caspase-3 activity inhibition (Fig. 4C–F). Similarly, Z-VAD tended to reduce liver cell injury in the I/R animal model (Fig. 4G–J, Supplementary Fig. 3C). The cell and animal experiments consistently showed increases in hepatocyte apoptosis and Caspase-3 activity during HIRI. The results suggest that RAMP1 inhibits hepatocyte apoptosis and promotes liver proliferation during HIRI.
RAMP1 protects hepatocytes against IRI by inhibiting the ERK/MAPK pathway and YAP phosphorylation
At 6 h after HIR, the number of cleaved Caspase-3–positive cells peaked and the phosphorylation of YAP was significantly enhanced, suggesting that apoptosis was the most severe. To identify the pathway that was significantly activated to cause increased apoptosis after RAMP1-KO, we used YAP and ERK phosphorylation inhibitors. CCK-8 revealed no significant effect on the activity of WT or RAMP1-KO primary hepatocytes under normoxic conditions (Fig. 5A). Under H/R, the YAP phosphorylation inhibitor PY-60 and ERK phosphorylation inhibitor SCH772984 significantly inhibited the H/R-induced apoptosis of RAMP1-KO primary hepatocytes (Fig. 5B). Other pathways, such as those of p-AKT and STAT3, did not significantly alter cell activity after inhibitor use under H/R. We detected changes in the MAPK pathway in WT and RAMP1-KO primary hepatocytes after H/R treatment. RAMP1-KO primary hepatocytes did not affect basic JNK, ERK1/2, or p38 signal transduction under control conditions (Fig. 5C–F). After 6 h of H/R treatment, the phosphorylation of JNK showed no significant change, that of p38 was significantly decreased, and that of ERK1/2 was significantly increased compared with those in the WT group. The phosphorylation of YAP is also shown in Figure 5C and G. Similar results were observed in WT and RAMP1-KO mice after HIRI, suggesting that RAMP1 affects apoptosis through ERK/MAPK and YAP (Fig. 5H–L).
RAMP1 protects hepatocytes against IRI by inhibiting the ERK/YAP pathway
The detection of apoptosis-related protein levels in primary hepatocytes after 6 h of H/R using the YAP phosphorylation inhibitor PY-60 and the ERK1/2 phosphorylation inhibitor SCH772984 showed different degrees of increased Bcl-2 expression and reduced Bax and cleaved Caspase-3 expression (Fig. 6A–C). These effects were also observed in the livers of RAMP1-KO mice (Fig. 6D–F). In the animal model, PY-60 and SCH772984 significantly reduced HIRI, protecting hepatocytes (Supplementary Fig. 4A–E). These results indicate that RAMP1 regulates HIRI-related apoptosis through ERK/MAPK and YAP.
Primary hepatocytes subjected to hypoxia and treated with PY-60 for 6 h showed reduced YAP phosphorylation, suggesting YAP pathway inhibition. However, the phosphorylation of ERK1/2 did not change; with SCH772984 administration during the sixth hour of H/R, the p-ERK1/2 level decreased as expected and YAP phosphorylation decreased significantly relative to the control (Fig. 6G). To elucidate the regulatory interplay among RAMP1, ERK1/2, and YAP, we employed the CGRP agonist CGRP II, rat TFA, known to efficaciously activate RAMP1 (Fig. 7A, B). Subsequently, CGRP agonists, an ERK inhibitor and agonist, and a YAP inhibitor and agonist were employed in the H/R model for CCK-8 analysis. CGRP agonist treatment substantially reduced H/R damage and increased cell activity, whereas the co-administration of a CGRP agonist with an ERK agonist (C16-PAF) or YAP inhibitor (verteporfin) significantly reduced cell activity. The co-administration of the CGRP agonist with SCH772984 or PY-60 increased cell activity (Fig. 7C, D), implying that RAMP1 acts upstream of YAP and ERK1/2. We administered SCH772984 with Verteporfin or PY-60 to scrutinize the regulatory relationship between ERK1/2 and YAP. SCH772984 and SCH772984 + PY-60 significantly boosted cell activity during H/R, whereas SCH772984 + Verteporfin notably reduced this activity. Conversely, C16-PAF + Verteporfin markedly reduced cell activity during H/R and C16-PAF + PY-60 increased this activity (Fig. 7E, F). These findings were corroborated by flow cytometry; the co-administration of the CGRP agonist with SCH772984 or PY-60 significantly reduced the proportion of apoptotic cells during H/R, whereas the co-administration of the CGRP agonist with C16-PAF or Verteporfin significantly increased this proportion (Fig. 7G, Supplementary Fig. 5A). Under H/R, SCH772984 + verteporfin and C16-PAF + Verteporfin substantially increased the proportion of apoptotic cells, whereas co-administration with PY-60 and C16-PAF + PY-60 significantly reduced this proportion (Fig. 7H, I, Supplementary Fig. 5B, C). These results strongly indicate that RAMP1 plays a role in the mitigation of hepatocyte injury by decreasing the phosphorylation of ERK1/2, which in turn leads to the reduction of YAP phosphorylation, during HIR.
Discussion
HIRI is a significant risk factor affecting survival after liver transplantation and contributing to donor shortages, and effective treatment strategies are urgently needed.41,42 This study was the first in which the role of the RAMP1 gene in mice subjected to HIR was investigated, and its results provide insight contributing to the identification of a novel therapeutic target for HIRI. It revealed that RAMP1 expression is upregulated significantly during HIRI, that the RAMP1 gene protects against HIRI by reducing hepatocyte apoptosis, and that the mechanism potentially underlying this hepatoprotective effect is related to the phosphorylation of YAP and the ERK/MAPK pathway.
In mice, RAMP1 deficiency leads to increased macrophage and mast cell infiltration of colon tissue and the elevation of TNF-α and IL-1β levels, and RAMP1-KO exacerbates dextran sulfate sodium–induced colitis.9 and increases inflammation, tissue edema, and pancreatic injury in the early stages of acute pancreatitis.10 RAMP1 has also been found to regulate the Hippo/Yap pathway and to promote CGRP-induced osteogenic differentiation of BMSCs.43 Its overexpression stimulates the proliferation of MSF through the Gαi3–PKA–CREB–YAP axis.11 Furthermore, RAMP1 deficiency severely impairs organ mass recovery and hepatocyte proliferation after acute and chronic liver injury.14 The protective effect of RAMP1 in different organs is supported by abundant evidence. Similarly, our recent studies have shown that RAMP1 plays a beneficial role under pathological conditions. H&E and TUNEL staining and liver function testing revealed aggravated liver tissue damage in RAMP1-KO mice relative to that in WT mice during IRI, but a minimal effect on RAMP1-KO liver tissue under physiological conditions, suggesting that RAMP1 helps to reduce the damage to liver structure and function during IRI. Given the critical role of RAMP1 in HIRI and its classification as a GPCR,8 these findings suggest that RAMP1 could serve as a therapeutic target for future drug development, paving the way for further clinical applications.
HIRI is characterized by apoptosis, but the underlying mechanism remains unclear.44 Several signal transduction pathways, including ERK/MAPK, have been found to play crucial roles in HIRI.19 The inhibition of the ERK/MAPK pathway has a protective effect during HIRI. Carbon monoxide (CO), for instance, inhibits the expression of early pro-inflammatory and stress response genes and effectively improves HIRI by activating the CO–MEK/ERK1/2 signaling pathway.45 Pretreatment with cafestol, a natural diterpene extract from coffee beans found mainly in unfiltered coffee, reduces ALT and AST levels, inhibits apoptosis, reduces the release of inflammatory mediators, and alleviates pathological liver damage, mainly by inhibiting ERK- and PPARγ-related pathways.46 Melatonin regulates the TLR-mediated inflammatory response and improves I/R-induced liver damage by blunting JNK and ERK phosphorylation.47 Cyclopamine pretreatment significantly reduces ERK phosphorylation and protects liver function after IRI.48 ERK1/2 plays a crucial role in the regulation of upstream factors contributing to apoptotic events by activating downstream transcription factors, inducing the release of cytochrome C, down-regulation of Bcl-2, and up-regulation of Bax. It also promotes apoptosis by activating and upregulating the expression of Caspases 3, 8, and 9.49,50 In this study, the subjection of RAMP1-deficient mice and cells to HIR significantly increased ERK activation and phosphorylation; treatment with SCH772984 inhibited ERK signaling, partially reversed the elevation of ERK phosphorylation, and reduced the rate of apoptosis. In addition, RAMP1-deficient mice exhibited significantly increased expression of pro-apoptotic proteins and decreased expression of anti-apoptotic genes compared with WT mice. These findings strongly suggest that RAMP1 plays a key role in the regulation of apoptosis in hepatocytes through the ERK/MAPK pathway.
YAP has been identified as a crucial component of the mammalian Hippo signaling pathway,51,52 and recent research has indicated it’s involvement in the regulation of apoptosis.53–56 For instance, YAP has been shown to play a role in glucose metabolism by promoting the expression of GLUT3, researchers have suggested that glucose starvation activates the Hippo–YAP signaling pathway, and YAP regulates apoptosis by controlling glucose uptake.57 Additionally, when normal blood cells experience DNA damage–related stress, the tyrosine kinase C-ABL translocates to the nucleus and phosphorylates YAP at Y357 on tyrosine residues.58 This phosphorylated YAP binds to p73 and promotes the transcription of pro-apoptotic genes such as p53AIPI,55 Bax,53 and PUMA.54 YAP interacts with TEAD transcription factors in the nucleus, leading to the upregulation of anti-apoptotic genes.59–61 Our findings suggest that the anti-apoptotic effect of RAMP1 in hepatocytes is related to the regulation of the ERK/MAPK pathway and the function and phosphorylation of YAP. RAMP1 contributes to the reduction of hepatocyte injury by diminishing the phosphorylation of ERK, leading to a decrease in YAP phosphorylation.
RAMP1 is a critical protein in CGRP receptor signaling, and RAMP1-KO has been demonstrated to impede CGRP function.13,14 Thus, we think that the mechanism underlying RAMP1’s regulation of HIRI involves the activation of CGRP, which, in turn, directly stimulates an increase in cAMP signaling. During I/R, the phosphorylation of ERK and YAP increases, leading to liver cell apoptosis and damage. CGRP-induced cAMP signaling may directly mitigate cell damage by inhibiting the phosphorylation of ERK and YAP. The findings of this study provide valuable insight into the biological role of RAMP1 and the mechanisms underlying RAMP1 damage in HIRI. RAMP1-targeted interventions may provide strategies for reducing IRI in various settings, including partial hepatectomy and liver transplantation.
Supporting information
Supplementary Table 1
The sequences of primer.
(DOCX)
Supplementary Fig. 1
Change in inflammatory cytokines in both WT and RAMP1-KO mice during HIRI.
(A–D) mRNA levels of inflammatory cytokines in the liver (n=3–5 per group) were assessed at different time points (n=3–5 per group). 18s rRNA served as a loading control for quantitative RT-PCR. All data are presented as the mean±SD. *p<0.05 compared with the sham groups; #p<0.05 compared with the WT + I/R groups using Student’s two-tailed t-test. TNF-α, tumor necrosis factor-α; IL-6/10/1β, interleukin-6/10/1β.
(TIF)
Supplementary Fig. 2
Change in apoptosis-related protein expression in both WT and RAMP1-KO mice during HIRI.
(A) and (B) Statistical analysis of pro-apoptotic and anti-apoptotic genes in WT and RAMP1-KO mice that underwent hepatic I/R (n=3–5 per group). (C) and (D) Statistical analysis of pro-apoptotic and anti-apoptotic genes in primary hepatocytes from WT and RAMP1-KO mice that underwent H/R (n=3–5 per group). All data are presented as the mean±SD. *p<0.05 compared with the sham groups; #p<0.05 compared with the WT I/R groups using Student’s two-tailed t-test. c-Casp-3, cleaved-Caspase3; Bax, bcl2-associated X protein; Bcl2, b-cell leukemia/lymphoma.
(TIF)
Supplementary Fig. 3
Electron micrograph of hepatocyte apoptosis in HIRI and effect of Z-VAD in vivo.
(A) Liver cells exhibiting pyknosis and shrunken characteristics, irregular nucleus with condensed chromatin, abundant mitochondria, dissolution of the matrix within the membrane across a large area, and focal cavitation; (B) Liver cells exhibiting slight pyknosis, the nucleus is irregular, nuclear membrane is intact, perinuclear space is widened (Red arrow), and increased heterochromatin; (C) HE of mouse livers after using Z-VAD under sham and I/R. Circles indicate necrotic areas. N, Nucleus; M, Mitochondria; RER, Rough endoplasmic reticulum; LD, Lipid droplets; I/R, ischemia/reperfusion.
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Supplementary Fig. 4
Effect of inhibitors on the model and co-localization of RAMP1 macrophages.
(A) HE and TUNEL staining was performed after PY-60 and SCH772984 treatment under hepatic I/R; (B) Necrosis area statistical analysis of HE. (C). TUNEL-positive cell statistical analysis. (D) ALT. (E) AST. (F) RAMP1 macrophage co-localization. The circle sign indicates the necrotic area. The red arrow indicates macrophages. All data are presented as the mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 using Student’s two-tailed t-test. HE, hematoxylin and eosin; TUNEL, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end-labeling; I/R, ischemia/reperfusion; ALT, alanine aminotransferase; AST, aspartate aminotransferase.
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Supplementary Fig. 5
Flow cytometry of regulatory interplay among RAMP1, ERK, and YAP during HIRI.
(A) Flow cytometry was used to detect the proportion of apoptosis in H/R with CGRP agonist, YAP inhibitor and agonist, and ERK inhibitor and agonist, respectively. (B) During H/R, ERK inhibitor combined with YAP inhibitor or agonist was used to detect the proportion of apoptosis using flow cytometry. (C) Under H/R, using ERK agonists combined with YAP inhibitors or agonists to detect the proportion of apoptosis, respectively. H/R, hypoxia/reoxygenation; ERK1/2, extracellular signal–regulated kinase1/2; YAP, yes-associated protein; p-YAP, phosphorated yes-associated protein; CGRP,caltonin gene-related peptide; VP, verteporfin.
(TIF)