Angiotensin II (Ang II) has been reported to aggravate hepatic fibrosis by inducing NADPH oxidase (NOX)-dependent oxidative stress. Alamandine (ALA) protects against fibrosis by counteracting Ang II via the MAS-related G-protein coupled (MrgD) receptor, though the effects of alamandine on hepatic fibrosis remain unknown. Autophagy activated by reactive oxygen species (ROS) is a novel mechanism of hepatic fibrosis. However, whether autophagy is involved in the regulation of Ang II-induced hepatic fibrosis still requires investigation. We explored the effect of alamandine on hepatic fibrosis via regulation of autophagy by redox balance modulation. In vivo, alamandine reduced CCl4-induced hepatic fibrosis, hydrogen peroxide (H2O2) content, protein levels of NOX4 and autophagy impairment. In vitro, Ang II treatment elevated NOX4 protein expression and ROS production along with up-regulation of the angiotensin converting enzyme (ACE)/Ang II/Ang II type 1 receptor (AT1R) axis. These changes resulted in the accumulation of impaired autophagosomes in hepatic stellate cells (HSCs). Treatment with NOX4 inhibitor VAS2870, ROS scavenger N-acetylcysteine (NAC), and NOX4 small interfering RNA (siRNA) inhibited Ang II-induced autophagy and collagen synthesis. Alamandine shifted the balance of renin–angiotensin system (RAS) toward the angiotensin converting enzyme 2 (ACE2)/alamandine/MrgD axis, and inhibited both Ang II-induced ROS and autophagy activation, leading to attenuation of HSCs migration or collagen synthesis. In summary, alamandine attenuated liver fibrosis by regulating autophagy induced by NOX4-dependent ROS.
Hepatic fibrosis is characterized by persistent deposition of extracellular matrix (ECM) components by hepatic stellate cell (HSC)-derived myofibroblasts. In the past two decades, HSCs have played an essential role in exploring mechanisms of hepatic fibrosis. The renin–angiotensin system (RAS) is involved in the development and pathogenesis of hepatic fibrosis [1–3]. As the pivotal bioactive peptide of the RAS, Angiotensin II (Ang II) has been recognized as a promoter of oxidative processes [4–6] and an inducer of autophagy [7–8] causes various cell dysfunctions and participates in disease states such as hypertension, cardiac remodeling, and organ fibrosis. The discovery of new components with biological activity in the RAS increases the complexity of this hormonal system. Some of these components (enzymes, intermediate products, and receptors) are described as having counter-regulatory activity against the classical angiotensin converting enzyme (ACE)/Ang II/Ang II type 1 receptor (AT1R) axis.
In 2013, a novel component of the RAS was defined by the Santos et al., was named as alamandine (Ala-Arg-Val-Tyr-Ile-His-Pro), and has very similar structure to Ang-(1–7) . The only difference is that in the N-terminal domain the amino acid alanine replaces aspartic acid. Alamandine (ALA) level is detectable in human plasma and elevated in patients with chronic renal failure, suggesting this molecule can exert biologically relevant actions [9,10]. Recently, actions of alamandine have been putatively linked to MAS-related G-protein coupled receptor (MrgD) activation [9,11]. The identification of two new components of the RAS, alamandine, and its receptor MrgD, will be important for improving our understanding of the physiological and pathophysiological role of this key regulatory system.
Angiotensin-(1–7) (Ang-(1–7)) is recognized as a hepatoprotective molecule and antagonist of Ang II actions in the liver. Previously, our research group has pointed out the protective role of Ang-(1–7) as a hepatic fibrosis regulator . Briefly, Ang-(1–7) suppresses NLRP3 inflammasome activation, improves oxidative stress, and attenuates Ang II-induced hepatocyte epithelial–mesenchymal transition (EMT) [4,12,13]. Since alamandine structure is highly similar to Ang-(1–7), their biological behavior seems likely to be closely related, although they act on different receptors. Cardiovascular actions of alamandine resemble actions observed for Ang-(1–7), such as long-lasting anti-hypertension and a decrease in fibronectin deposition [9,14–16]. Currently, however, little is known concerning alamandine function in fibrotic disease in general and hepatic fibrosis in particular.
Macrophagocytosis (referred to as autophagy) is a vacuolar, self-digesting mechanism for sequestration, and subsequent degradation of long-lived proteins and impaired organelles by lysosomes. Autophagy is involved in many human fibrotic diseases [7,17–19]. Recent studies show that HSC autophagy can promote fibrosis, which depends on its direct contribution to the activation process of HSC by providing energy substrates [20–22]. Various pathways have been proposed to regulate autophagy, among which reactive oxygen species (ROS) represent one of the most important activators [23,24]. NADPH oxidase (NOX) 4 (NOX4) is a non-phagocytic homologous form of NOX, which constitutively produces hydrogen peroxide (H2O2) and ROS, promoting the critical event in the process of hepatic fibrosis [4,25,26]. The majority of existing evidence indicates that NOX4 plays an important role in autophagy regulation [27–29]. Our previous research shows that Ang II treatment increased the expression of NOX4 protein and ROS production in HSCs  and Ang II has been considered as an inducer of autophagy [30–32]. Thus, alamandine could improve liver fibrosis by inhibiting Ang II-induced NOX4-dependent ROS and subsequent activation of autophagy.
The present study aimed to investigate the ability of the alamandine/MrgD axis to counter-regulate Ang II actions in HSCs. We discuss the differential effects of the two RAS axes on autophagy via NOX4-dependent ROS in hepatic fibrosis, and demonstrate that alamandine attenuates hepatic fibrosis by regulating redox balance and autophagy in vivo and in vitro.
Materials and methods
Ang II, Ang-(1–7), A779, 3-methyladenine (3MA), and rapamycin were purchased from Sigma–Aldrich (St. Louis, MO). Alamandine was purchased from Biosyntan (Berlin, Germany). D-Pro7-Ang-(1–7) was purchased from Bachem (Torrance, U.S.A.). β-alanine was purchased from Capotchem (HangZhou, China). VAS2870 and N-acetylcysteine (NAC) was provided by SelleckChem (Houston, TX). MrgD small interfering RNA (siRNA), NOX4 siRNA, and microtubule-associated protein1A/1B-light chain 3 (LC3) siRNA were provided by GenePharma (Shanghai, China). Other reagents are described below.
Animal experimental design
Sprague–Dawley (SD) rats were provided by the laboratory animal center and the study protocol was approved by the Animal Experiment Ethics Committee of the Southern Medical University. Animals were maintained in a specific pathogen-free facility (Central Animal Care Facility of Southern Medical University, Guangzhou, China), under controlled environment (12-h light/dark cycle; temperature, 22–24°C) and were provided with standard food and water ad libitum.
A rat model using carbon tetrachloride (CCl4)-induced liver fibrosis was used. We established six groups using male SD rats (180–200 g): vehicle group (n=10), CCl4 group (n=10), alamandine group (n=10), CCl4+alamandine group (n=10), CCl4+3MA group (n=10), and CCl4+rapamycin group (n=10). The vehicle group received an intraperitoneal injection of olive oil, 2 ml/kg, twice a week. The four CCl4 groups received an intraperitoneal injection of a 40% CCl4-olive oil solution, 2 ml/kg body weight, twice a week. The alamandine group was subcutaneously implanted with micro-osmotic pumps to permit continuous infusion with alamandine at a rate of 25 µg/kg.h. The 3MA-treated group was treated with 10 mg/kg 3MA per day, whereas the rapamycin group was treated with 1 mg/kg rapamycin per day. Rats were killed to obtain liver tissue and serum for a trial scheduled at the end of the 4th week. A total of 1.5–2% isoflurane was used during the implantation of the micro-osmotic pumps. Euthanasia was done under deep anesthesia using 5% isoflurane inhalation and maintained throughout the surgical procedure.
Histological analysis and immunohistochemistry
Liver specimens were incubated with the following primary antibodies: a-smooth muscle actin (a-SMA; 55135-1-AP; 1:200, Proteintech, Rosemont, U.S.A.), COL1A (α-collagen I; ab93095; 1:200, Abcam, Cambridge, U.K.), NOX4 (ab133303; 1:200, Abcam, Cambridge, U.K.), LC3B (ab51520; 1:200, Abcam, Cambridge, U.K.), P62 (ab56416; 1:200, Abcam, Cambridge, U.K.), and then incubated with streptavidin–biotin. Peroxidase conjugates were visualized with diaminobenzidine reagent.
Cell isolation, identification, culture
The preparation and culture of primary rat HSCs from the livers of male SD rats were separated by collagenase/proenzyme perfusion and Nycodenz gradient as previously described . The HSCs were identified by flow cytometry and immunofluorescence cytochemistry. HSCs were cultured in Dulbecco’s modified Eagle’s medium (DMEM; GIBCO BRL, Life Technologies, Carlsbad, CA) with 20% FBS (GIBCO BRL, Life Technologies, Carlsbad, CA) at 37°C in 5% CO2 incubator.
Measurement of hepatic hydroxyproline content
The content of hydroxyproline in liver was measured using hydroxyproline detection kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions; absorbance was measured at 550 nm.
Cell migration was evaluated using a transwell assay with the cell culture inserts (#3422; Corning, New York, U.S.A.). Briefly, The upper chamber of a transwell apparatus contained cells (1 × 105 cells/ml serum-reduced medium), while the lower chamber contained medium alone. Cells were incubated at 37°C for 24 h. Migrant cells on the lower surface of the membranes were fixed and stained with Jimsar dyeing. Migration was evaluated by counting the number of cells that moved through the transwell chambers. All experiments were repeated at least thrice.
The H2O2 concentration was detected using Hydrogen Peroxide Assay Kit (Beyotime, Shanghai, China). Briefly, the cells were cracked in lysis buffer, followed by the addition of H2O2 detection solution, supernatant, and standard substance. Liver tissue (10 mg) was homogenized in 150 μl lysis buffer, and then centrifuged at 4°C, 12000×g for 5 min. Supernatant (50 μl) was incubated with reaction solution (100 μl) under at 25°C for 30 min. Finally, optical density was measured at 560 nm. A standard concentration curve was used to calculate the release concentration of H2O2 through triplicate experiments.
Intracellular ROS detection
The generation of ROS was assessed in primary HSCs using 2′,7′-dichlorofluorescein diacetate (H2-DCFDA; Molecular Probes, Life Technologies, NY, U.S.A.) probe, which is an ROS-sensitive membrane-permeable fluorescent probe. DCFH can be oxidized to fluorescent 2′,7′-dichlorofluorescein (DCF) by ROS. After exposure to a stimulus, cells were incubated with different concentrations of pterostilbene for 8 h, then exposed to 10 mM of DCFH-DA at 37°C for 30 min. DCFDA fluorescence was quantified using a multiwell fluorescence scanner (Spectra Max M5/M5e, Molecular Devices, U.S.A.). Fluorescence was measured with 480-nm excitation and 525-nm emission wavelengths. Images were analyzed using FV10i-ASW 3.0 Viewer software. All experiments were repeated at least thrice.
Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured in serum using biochemical kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer’s instructions. All experiments were repeated at least thrice.
Monitoring HSCs autophagic flux
Primary rat HSCs were transfected with an mRFP-GFP-LC3 encoding plasmid (Hanbio, LP2100001). Transfection efficiency was 70%. Autophagic flux was visualized using a confocal microscopy (Fluoview FV10i, Olympus, Japan). All experiments were repeated at least thrice.
Cells were fixed with 2.5% glutaraldehyde, dehydrated, and coated gold in the coating apparatus. Autophagosomes and autolysosomes were observed under the transmission electron microscope (TEM) at an 80-kV acceleration voltage. All experiments were repeated at least thrice.
The following siRNA sequences of targeted proteins were used: NOX4 (sense: 5′-CCGGACAGTCCTGGCTTATC-3′; antisense: 5′-GGCTACATGCACACCTGAGA-3′); MrgD (sense: 5′-TGGCAGAGAGGTGGAGTGTA-3′; antisense: 5′-GCACATAGACACAGAAGGGAGA-3′); LC3B (sense: 5′-ATGGGGAAAAGCACGGAGAG-3′; antisense: 5′-UUCAUCUGCCUGCUUGUCCTT-3′). All experiments were repeated at least thrice.
RNA isolation and quantitative real-time polymerase chain reaction
The following primers were used: ACE primer (sense: 5′-CTTGACCCTGGATTGCAGCC-3′; antisense: 5′-CAGAGAGTTGTACTGCAGCCG-3′); AT1R primer (sense: 5′-TCTGCCACATTCCCTGAGTTA-3′; antisense: 5′-TCACCACCAAGCTGTTTCCA-3′); ACE2 primer (F: 5′-ACAATTGTTGGAACGCTGCC-3′; R: 5′-CGATCTCCCGCTTCATCTCC-3′); MrgD primer (F: 5′-TTTTCAGTGACATTCCTCGCC-3′; R: 5′-GCACATAGACACAGAAGGGAGA-3′). All experiments were repeated at least thrice.
The primary antibodies were MrgD (ab240132; 1:1000, Abcam, Cambridge, U.K.), NOX4 (ab133303; 1:1000, Abcam, Cambridge, U.K.), connective tissue growth factor (CTGF; ab6992; 1:1000, Abcam, Cambridge, U.K.), COL1A (ab93095; 1:1000, Abcam, Cambridge, U.K.), LC3B (ab51520; 1:1000, Abcam, Cambridge, U.K.), P62 (ab56416; 1:1000, Abcam, Cambridge, U.K.), AMPK (AMP-dependent protein kinase; ab32047; 1:1000, Abcam, Cambridge, U.K.), unc-51 like autophagy activating kinase 1 (ULK1; ab203207; 1:1000, Abcam, Cambridge, U.K.), α-SMA (55135-1-AP; 1:1000, Proteintech, Rosemont, U.S.A.), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 60004-1-Ig; 1:7500, Proteintech, Rosemont, U.S.A.). The secondary antibodies were donkey anti‐mouse and goat anti-rabbit (1:7500; LI-COR Bioscience, Lincoln, NE). Immunoblot detection was performed using chemiluminescence with an Odysseys Infrared Imaging System. All Western blots were repeated at least thrice.
Data are expressed as means ± standard deviation. A two-tailed Student’s t test was used for statistical analysis of two groups. ANOVA analyses using SPSS 22.0 software were utilized when more than two groups were assessed. Statistical P-values <0.05 were considered significant.
Constant infusion with alamandine attenuates oxidation, autophagy, and hepatic fibrosis induced by CCl4 in rats
To explore the precise mechanism of hepatic fibrosis, CCl4-induced liver fibrosis in an animal model was established. Sections of rat liver tissue from CCl4 group rats, stained with Hematoxylin and Eosin, displayed severe liver fibrosis compared with vehicle group animals. Constant infusion with alamandine improved CCl4-induced liver fibrosis. Similarly, hydroxyproline content assay and Masson’s trichrome staining showed more collagen deposition in CCl4 group animals with higher Metavir scores and Ishak scores. In contrast, CCl4+alamandine group showed less collagen deposition and lower scores (Figure 1A,B,D,E). Immunohistochemical staining indicated that COL1A and a-SMA protein levels increased in fibrotic liver induced by CCl4 and that alamandine treatment counteracted this build up (Figure 1C). Moreover, treatment with alamandine inhibited the increase in α-SMA, COL1A, and CTGF protein levels in CCl4 group animals (Figure 1F). Finally, alamandine reduced levels of ALT and AST in serum (Figure 1G). Thus, alamandine attenuated CCl4-induced liver fibrosis.
Constant infusion with alamandine attenuates the oxidation levels, autophagy, and liver fibrosis induced by CCl4 in rats
Next step, we assessed the level of oxidation in liver tissue. NOX4 protein levels were increased in liver from CCl4 group rats. This increase was suppressed by alamandine treatment (Figure 1C). Similarly, H2O2 content was markedly increased in CCl4 group animals, and this effect was also reversed by treatment with alamandine (Figure 1H). Hence, alamandine improved CCl4-induced liver fibrosis via inhibiting oxidative stress.
Finally, we used rapamycin to raise autophagy status and used 3MA to lower autophagy status in CCl4-treated liver tissues. Hydroxyproline content assay and Masson’s trichrome staining revealed severe liver fibrosis in the CCl4 plus rapamycin group rats with higher Metavir scores and Ishak scores compared with CCl4 group animals. In CCl4 plus 3MA group, CCl4-induced liver fibrosis was mitigated (Figure 1A,B,D,E). Western blot analysis validated the conclusion that inhibiting autophagy reduced collagen deposition (Figure 1F). Moreover, in CCl4 plus rapamycin group animals, higher NOX4 protein levels and the H2O2 content were induced by pretreatment with rapamycin. The opposite results, attenuation of autophagy with 3MA or alamandine treatment, suggests that inhibiting autophagy could suppress oxidative stress induced by CCl4 (Figure 1C,H).
Additionally, immunohistochemical staining was used to demonstrate autophagy status in liver tissues. Accumulation of P62 and LC3 might indicate that autophagy has a role in the pathogenesis of liver fibrosis, and autophagy inhibitors such as 3MA could alleviate CCl4-induced liver fibrosis. Continuous subcutaneous injection of alamandine could also counteract the enhancement induced by CCl4 (Figure 1C). This effect was confirmed by Western blot analysis (Figure 1F). Finally, inhibiting autophagy could suppress the increase in ALT and AST levels in serum (Figure 1G).
These results suggested that alamandine counteracted CCl4-induced liver fibrosis. The mechanism for this action might be related to suppressing oxidative stress levels and consequent autophagy.
Alamandine reduces Ang II-induced HSC activation and collagen production
In vitro, Ang II-induced HSC activation and collagen production was reduced by alamandine treatment. Levels of COL1A, α-SMA, and CTGF proteins were decreased by alamandine treatment in a concentration- and time-dependent manner (Figure 2A,B). Further, we compared the effects of alamandine and Ang-(1–7) on HSC activation and collagen production in rat HSCs. Both showed the same effect on reducing collagen synthesis (Figure 2C). However, treatment with the MAS receptor antagonist A-779 failed to block alamandine’s anti-fibrosis effects following induction by Ang II (Figure 2D).
Alamandine reduces Ang II-induced HSC activation and collagen production
Alamandine exerts anti-fibrotic actions via the MrgD receptor along with up-regulation of the ACE2/alamandine/MrgD axis in HSC
Locally based RAS is involved in the development of liver fibrosis. Accumulating evidence shows that up-regulation of the ACE/Ang II/AT1R axis exacerbates liver fibrosis. A recent study found that the ACE2/alamandine/MrgD axis counters the activity of the ACE/Ang II/AT1R axis. Thus, up-regulation of the ACE2/alamandine/MAS axis may prevent Ang II-induced liver fibrosis and we investigated the influence of Ang II and alamandine on the balance of these two axes in HSC. Expression levels of RAS components, including ACE, AT1R, ACE2, and MrgD, were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot (Figure 3A,C–F). Results indicated that Ang II decreased ACE2 and MrgD protein levels and increased ACE and AT1R protein levels, thus shifting the balance toward the ACE/Ang II/AT1R axis. Conversely, alamandine increased ACE2 and MrgD levels and decreased ACE and AT1R protein levels, moving the balance from the ACE/Ang II/AT1R axis toward the ACE2/alamandine/MrgD axis. The ACE2/alamandine/MrgD axis counteracts the profibrotic effects of the ACE/Ang II/AT1R axis.
Alamandine exerts anti-fibrotic actions via MrgD receptor in HSCs along with up-regulation of the ACE2/alamandine/MrgD axis
Ang II-induced COL1A, α-SMA, and CTGF protein levels could be constrained by alamandine, while the MrgD receptor antagonist D-Pro7-Ang-(1–7) reversed the effects. β-alanine, a MrgD agonist, prevented Ang II-induced HSC activation and collagen production, indicating that MrgD receptors exert anti-fibrotic actions (Figure 3G). In addition, MrgD siRNA significantly inhibited alamandine’s anti-fibrotic actions and β-alanine did not prevent Ang II-induced collagen synthesis after MrgD receptor depletion by MrgD siRNA in HSCs (Figure 3H,I). These data suggest that alamandine plays its biologic role primarily via the MrgD receptor, independent of the MAS receptor.
Ang-(1–7) alone or in combination with alamandine up-regulated expression of MrgD (Figure 3B). Moreover, Ang II-induced increases in COL1A, α-SMA, and CTGF protein levels could be inhibited by Ang-(1–7) after MAS receptors are depleted by MAS siRNA in HSCs, and the MrgD receptor antagonist D-Pro7-Ang-(1–7) reversed these effects (Figure 3H,J). These results suggest that MrgD receptor could be activated by Ang-(1–7).
Alamandine suppresses AngⅡ- induced collagen production by suppressing oxidative stress in HSCs
Ang II promoted NOX4, COL1A, α-SMA, and CTGF protein levels. Alamandine significantly inhibited the increases, and these inhibitory effects were reversed by the MrgD receptor antagonist D-Pro7-Ang-(1–7). Pretreatment with NAC or VAS2870 inhibited the increase in NOX4, COL1A, α-SMA, and CTGF protein levels induced by Ang II treatment (Figure 4A). Analogously, Ang II-induced ROS and H2O2 production were inhibited by alamandine, and D-Pro7-Ang-(1–7) reversed these effects. Ang II-induced intracellular ROS and H2O2 accumulation were suppressed by NAC or VAS2870 (Figure 4D,E). Further, results show that NOX4 siRNA and MrgD siRNA suppressed Ang II-induced HSC activation and collagen production (Figure 4B,C). The number of migrating cells was increased by Ang II and decreased by pretreatment with alamandine, VAS2870, and NAC (Figure 4F). These data confirmed that alamandine inhibited Ang II-induced HSC activation and collagen synthesis via inhibiting oxidative stress.
Alamandine suppresses Ang II-induced collagen production by inhibiting oxidative stress in HSCs
Alamandine attenuates AMPK-dependent autophagy in HSCs
Emerging evidence demonstrates that autophagy partially exacerbated formation of liver fibrosis, which leads to the question of the influence of autophagy on collagen genesis. We examined the effects of Ang II and alamandine on autophagy in rat HSCs. Levels of AMPK, ULK1, P62, and LC3BII/I proteins showed that alamandine treatment attenuated AMPK-dependent autophagy, which was enhanced by Ang II, in a concentration- and time-dependent manner (Figure 5A–D). Exposure to bafilomycin A1 (BA) increased collagen deposition in HSCs (Figure 5E). Pretreatment with alamandine suppressed the activation of autophagy induced by Ang II treatment (Figure 5F,H). Using LC3B siRNA, we confirmed that collagen expression was reduced in response to LC3B siRNA (Figure 5G). These results indicated that Ang II-induced AMPK-dependent autophagy enhanced collagen synthesis, but such action was reduced by alamandine treatment.
Alamandine attenuates the AMPK-dependent autophagy in HSCs
Alamandine suppresses Ang II-induced collagen production by regulating NOX4/ROS/autophagic flux
Alamandine inhibited Ang II-induced LC3B enhancement and this inhibitory effect was reversed by D-Pro7-Ang-(1–7) (Figure 6A). Pretreatment with NOX4 siRNA neutralized Ang II-induced LC3B conversion (Figure 6B). This finding suggested that NOX4-generated ROS may be needed to activate autophagy. To evaluate autophagic flux, we used adenovirus-harboring mRFP-GFP-LC3. After pretreatment with alamandine, fewer numbers of GFP and mRFP dots per cell were observed compared with Ang II treatment alone. In addition, fewer yellow dots were seen in merged images of tissue from Ang II + alamandine group rats, indicating that alamandine partially ameliorated Ang II-induced sufficient autophagic flux (Figure 6C). However, the protective effect of alamandine was neutralized by D-Pro7-Ang-(1–7). Further, Ang II-induced autophagic flux was attenuated by the NOX4-targeted inhibitor VAS2870, suggesting that alamandine suppressed AngⅡ-induced collagen production by regulating the NOX4/ROS/sufficient autophagic flux (Figure 6C).
Alamandine suppresses Ang II-induced collagen production by regulating the NOX4/ROS/autophagic flux
The main finding of the present study was that we have first demonstrated that alamandine attenuates hepatic fibrosis by regulating autophagy caused by NOX4-generated ROS (Figure 7). Principal findings include: (i) alamandine regulates the balance of the RAS from the ACE/Ang II/AT1R axis to the ACE2/alamandine/MrgD axis in HSC, (ii) AMPK-dependent autophagy is activated by Ang II-induced NOX4-derived ROS, and (iii) alamandine ameliorates hepatic fibrosis by regulating redox balance and autophagy in vivo and in vitro.
A schematic view of major signal transduction pathways involved in alamandine attenuates CCl4-induced liver fibrosis via inhibiting autophagy caused by NOX4-dependent ROS
In the present study, we focused on a novel angiotensin, alamandine, and its putative receptor, MrgD. Alamandine can be generated from either ‘deleterious’ angiotensin A (Ang A) or ‘protective’ Ang-(1–7). Alamandine has been identified as a product of the catalytic hydrolysis of the octopeptide Ang A by ACE2; futhermore, this peptide can be produced directly from Ang-(1–7) through decarboxylation of N-terminal aspartate amino acid residue [9,11]. Considering that alamandine has a similar structure to Ang-(1–7), the possibility that alamandine may bind to the MrgD receptor or to the Ang-(1–7) receptor MAS was tested. The present data demonstrate protective action of alamandine in liver fibrosis. Our results show that alamandine inhibited HSC activation and collagen production independently from the MAS receptor. Unlike Ang-(1–7), treatment with the MAS receptor antagonist A-779 did not block alamandine anti-hepatic fibrotic action. The possibility of MAS as the primary binding site of alamandine is, thus, ruled out.
Next, we investigated the possible interaction between alamandine and MrgD. The anti-hepatic fibrotic effects of alamandine could be inhibited by the MrgD antagonist D-Pro7-Ang-(1–7) and the MrgD ligand β-alanine, indicating that alamandine counters Ang II via the MrgD receptor. Further, MrgD siRNA alleviated Ang II-induced collagen deposition in HSCs. These results are of major importance since they strongly support a key anti-fibrotic role for MrgD receptors in HSCs and further strengthened confidence in the conclusion that alamandine induces protective signaling via MrgD. In summary, the study of alamandine and its receptor MrgD might lead to a new understanding of RAS physiology and reveal possibilities for novel therapeutic strategies for liver diseases.
Oral administration of alamandine has an anti-fibrotic effect in isoproterenol-treated rats and an anti-hypertensive effect in SHR . In other work, alamandine shows anti-fibrotic and anti-inflammatory effects in mouse aorta . Previously, we have found that Ang-(1–7) exerts a hepatic anti-fibrotic action against Ang II through the regulation of the redox balance and subsequent NLRP3 inflammasome activation . In our study, both alamandine and Ang-(1–7) have anti-fibrotic effects in HSC, despite activating distinct receptors. The finding that alamandine induces a similar effect raises the intriguing question of whether these two peptides exert additive actions in HSCs. This possibility deserves further study. However, not all actions displayed by alamandine are similar to Ang-(1–7) . For example, alamandine and Ang-(1–7) have opposing effects on the expression and secretion of leptin in adipose tissue . It is possible that the action of MAS and MrgD in adipocytes exhibit opposite actions.
Recent findings regarding the interaction of alamandine and Ang-(1–7) with MrgD receptors reveal that the role of MrgD-mediated signaling is more convoluted than suspected in the RAS. MrgD can be activated by Ang-(1–7) signaling, and this signaling cascade involves to protein kinase A, cAMP, and adenylyl cyclase [37–38]. In agreement, our results showed that Ang-(1–7) alone, or in combination with alamandine, up-regulate expression of MrgD. Along with interfering with MAS siRNA, Ang-(1–7) could still exert anti-fibrotic actions via MrgD receptor. MrgD thus appears to serve as a dual receptor. This finding may explicate why some reports have described a blockade of some Ang-(1–7) effects with D-Pro7-Ang-(1–7) but not with A-779 [39–41]. Note that MrgD as alamandine receptor does not exclude the possibility of other binding sites, such as the MrgE receptor . Ava et al. found that alamandine can increase blood pressure by activating AT1R under physiological conditions, while it can reduce blood pressure by activating AT2R or MrgD receptors under pathological conditions . In this context, differential receptor expression patterns can assure more effective therapeutic efficacy for these peptides for certain pathophysiological states. Whether binding sites other than MrgD are biologically relevant for alamandine actions in the liver is an open question and deserves further attention.
RAS represents an ingenious system of ‘checks and balances’. On the one hand it contains vasoconstrictive, pro-proliferative, and pro-fibrogenic compounds; on the other hand, it includes molecules with the opposite effects. Up-regulation of ACE/Ang II/AT1R axis in organ fibrosis, including the increased level of ACE, Ang II, and AT1R, contributes to the pathogenesis of fibrosis [34,44–46]. The RAS also plays an essential role in the development of hepatic fibrosis. Our data indicate that Ang II treatment not only exacerbates oxidative stress, autophagy, and collagen deposition in HSCs, but also shifts the balance to the ACE/Ang II/AT1R axis. Previous reports show that enhanced activity of the ACE/Ang II/AT1R axis is associated with fibrogenesis and steatosis in liver and ACE2/Ang-(1–7)/MAS plays the opposite role . The ACE2/Ang-(1–7)/MAS axis of the RAS is an essential counter-regulatory mechanism for the ACE/Ang II/AT1R axis. Interestingly, alamandine inhibits Ang II-induced hepatic fibrosis and shifts the balance to the ACE2/alamandine/MrgD axis. The ACE2/alamandine/MrgD axis could be another protective axis similar to the ACE2/Ang-(1–7)/MAS axis, to counterbalance the ACE/Ang II/AT1R axis. Up-regulation of the ACE2/alamandine/MrgD axis may be a promising strategy for treatment of liver fibrosis.
The pathogenesis of hepatic fibrosis is an intricate process involving many cell types . Among them, myofibroblasts, which derive from HSCs, are considered as primary effector cells in liver fibrosis . Autophagy is a new, but convoluted, regulatory pathway for hepatic fibrosis. Growing evidence emphasizes a dual role for autophagy in the function and phenotype of intrahepatic cells, depending on the type and stage of cells. On one hand, autophagy prevents hepatocyte apoptosis and maintains macrophages from impairment to improve liver fibrosis and injury [49,50]; on the other hand, autophagy also induces liver sinusoidal endothelial cell (LSEC) defenestration [51,52] and biliary epithelial cells senescence that aggravate hepatic fibrosis . Recent findings showing that the protective effect of autophagy induction on hepatocyte damage and hepatic fibrosis is in sharp contrast with the profibrogenic effects of autophagy in HSCs. In the present study, multiple approaches were used to demonstrate that Ang II stimulation promotes autophagy activity through the AMPK/ULK pathway during HSC activation, as assessed by the increase in LC3B II/I ratio and autophagic flow. Electron microscopy, as the gold standard for autophagy, also confirmed this result. Both in vivo and in vitro, LC3B expression was remarkably increased together with collagen synthesis. Moreover, Ang II-induced HSC activation and collagen deposition could be ameliorated by LC3 siRNA. Notably, alamandine suppresses autophagy activation and ameliorates Ang II-induced HSC activation and collagen deposition. Further study is needed to understand how alamandine induces this response.
Oxidative stress, an imbalance between excessive ROS production and antioxidant capacity of cells, is considered the main cause of liver fibrosis [54–56]. As a stimulus to autophagy, ROS is involved in various signaling pathways of autophagy activation. Excessive intercellular ROS is mainly produced by mitochondria and NOX. The majority of existing evidence indicates that NOX4 plays a major role in the control of autophagic machinery that depends on ROS production [27,57,58]. Our study found that pretreatment with NOX4 siRNA, ROS scavenger NAC, and NOX4 inhibitor VAS2870 dramatically reduced protein levels of LC3B-II, highlighting the important role of NOX4-generated ROS in the course of autophagosome accumulation. Moreover, the antioxidant and NOX4 inhibitor has inhibitory effects on autophagic flux. Thus, we hypothesized that alamandine infusion alleviates liver fibrosis by inhibiting NOX4-dependent ROS and subsequent activation of autophagy.
For the first time, our study shows the protective role of alamandine in liver fibrosis. In vivo, constant infusion with exogenous alamandine significantly down-regulated oxidative stress and reduced autophagy, which resulted in amelioration of liver fibrosis in CCl4-treated rats. In vitro, alamandine inhibited autophagy by suppressing NOX4-derived ROS.
In conclusion, we demonstrate that alamandine attenuates liver fibrosis by attenuating the activation of autophagy induced by NOX4-dependent ROS. However, the signaling mechanism by which ROS regulates autophagy have rarely been clarified. Current studies indicate that ROS can influence autophagy through different pathways depending on generation source, ROS species, and production. Classical pathways include PI3K-Akt-mTOR and AMPK-mTOR; other important molecules are also involved, such as Atg4, Foxo, and JNK [59–62]. In addition, recent evidence suggests that ROS can indirectly regulate autophagy through the control of the ubiquitin–proteasome system [28,63,64].
Signaling from AMP-activated protein kinase (AMPK), a key nutrient-sensitive kinase, is important for nutritional sensing of the autophagy pathway. AMPK subunits were found to interact with Unc-51-like kinase 1 (ULK1) in the autophagy interaction network and were independently verified as binding partners of ULK1 . AMPK enhances autophagy by directly phosphorylating and activating ULK1 kinase at Ser317 and Ser777 [66–68]. As previously reported, alamandine, via MrgD, induces AMPK/NO signaling to counter-regulate ANG II-induced hypertrophy . Our findings show that alamandine alleviates Ang II-induced autophagy through inhibiting ULK1 phosphorylation by AMPK.
The study has some limitations. As a potential therapeutic target for hepatic fibrosis, the effects of alamandine were not evaluated in patients suffering from this condition. Future research will focus on the therapeutic effect of almandine for patients with hepatic fibrosis.
In conclusion, we demonstrate for the first time a beneficial effect of alamandine against liver fibrosis through the inhibition of NOX4-derived ROS and subsequent autophagic activation. Consequently, the present study opens new avenues for the use of alamandine as a promising therapeutic target for treating liver fibrosis.
Hyperactivity of ACE/Ang II/AT1R, the classical axis of the RAS, is involved in the development and pathogenesis of hepatic fibrosis.
Recent studies show that alamandine, a novel component of the RAS, has anti-hypertensive, anti-fibrotic, and anti-inflammatory effects.
The present study shows that the ACE2/alamandine/MrgD axis represents an important hepatoprotective counter-regulatory mechanism within the RAS.
Results reveal a new mechanism of alamandine attenuation of liver fibrosis, suggesting that alamandine is a promising target for treatment and prevention.
The authors declare that there are no competing interests associated with the manuscript.
This work was supported by the National Natural Science Foundation of China [grant numbers 81670556, 81873583].
Y.H. (Yun Huang) contributed to design, data acquisition and analysis, and drafted the manuscript. A.L. and Y.L. (Yang Li) contributed to data acquisition, analysis, interpretation. G.-z.W., Y.H. (Ye Hu), Y.Z., and W.H. contributed to data acquisition and analysis. J.W., Y.L. (Yue Li) and X.Z. contributed to data acquisition. T.C. and J.L. contributed to data analysis. Y.M. and X.L. contributed to conception, design, data acquisition, analysis, and interpretation and drafted and critically revised the manuscript.
The authors are much grateful to Prof. Pingsheng Wu, Prof. Zhenshu Zhang, and Prof. Xishan Yang for essential help in the present study.
angiotensin converting enzyme 2
AMP-dependent protein kinase
- Ang II
a/α-smooth muscle actin
angiotensin II type 1 receptor
connective tissue growth factor
hepatic stellate cell
microtubule-associated protein1A/1B-light chain 3
MAS-related G-protein coupled receptor
reactive oxygen species
small interfering RNA
unc-51 like autophagy activating kinase 1
These authors contributed equally to this work.