Corin inhibits microglial inflammatory activation by suppressing mitochondrial dysfunction in intracerebral hemorrhage

Abstract

Microglial activation driving neuroinflammation is a key factor in secondary brain injury after intracerebral hemorrhage (ICH); however, the regulatory mechanisms remain unknown. This study investigates how corin influences microglial inflammatory activation and its underlying mechanisms. Corin expression in rat ICH brain tissue was assessed at multiple time points. To assess corin’s effect on neurological function and microglial inflammation, ICH rats and oxygen–glucose deprivation plus hemin (OGD/H)-stimulated HAPI microglia were treated with corin-encoding lentivirus. Neurobehavioral performance was evaluated using the Morris water maze (MWM). Microglial activation was assessed via Iba-1, iNOS, and Arg-1 expression, cytokine secretion, and migration assays. To determine whether AMPK mediates corin’s effects, cells were co-treated with Compound C (an AMPK inhibitor) and corin lentivirus. Corin expression decreased in ICH rat brain tissue, reaching its lowest level on day 3 post-ICH. Corin overexpression protected against ICH-induced neuronal apoptosis and improved neurological deficits as confirmed by MWM. Moreover, corin overexpression reduced microglial activation and inflammation in both ICH rats and OGD/H-stimulated microglial cells, ameliorated mitochondrial dysfunction, and increased the p-AMPK/AMPK ratio. The protective effects of corin on cell migration, inflammation, and mitochondrial function were reversed by Compound C, indicating AMPK is a downstream mediator of corin. Targeting corin offers a promising therapeutic strategy for ICH by reducing neuroinflammation and mitochondrial damage through AMPK activation and modulation of microglial inflammatory phenotype polarization.

INTRODUCTION

Intracerebral hemorrhage (ICH), a severe stroke subtype accounting for approximately 10%-20% of all strokes, has multiple risk factors including male sex, advanced age, diabetes mellitus, chronic kidney disease, and cerebral microbleeds (1). Beyond the immediate damage caused by hematoma formation and mass effect—compressing brain parenchyma and causing structural disruption—ICH triggers secondary neurological deterioration. This phase involves microglia-driven neuroinflammation characterized by release of neurotoxic mediators, blood–brain barrier disruption, vasogenic edema, and apoptosis of neurons and glial cells (2, 3). As primary innate immune responders to extravasated blood (4), microglia activate and polarize into pro- and anti-inflammatory phenotypes. They secrete cytokines, chemokines, and proteases, facilitating post-injury repair (5, 6). In ICH, activated microglia exacerbate neuroinflammation by recruiting circulating immune cells and releasing cytotoxic factors that stimulate lymphocytes, creating inflammatory cycles, increasing perilesional blood–brain barrier permeability, and inducing vasogenic edema (7). Therefore, targeting microglial neuroinflammation is crucial for improving ICH outcomes (8, 9). Understanding the molecular mechanisms underlying microglial inflammatory activation in ICH is essential to identify therapeutic targets to reduce post-ICH inflammation.

Mitochondria regulate immune cell function through metabolic processes like ATP production and biosynthesis (10). Microglial mitochondrial dysfunction worsens neuroinflammation, particularly in Alzheimer’s disease (11, 12). In murine ICH models, mitochondrial transplantation reduces proinflammatory microglial polarization, restores immune balance, and improves neuromotor deficits (13). Thus, targeting mitochondria is a vital strategy for controlling neuroinflammation in ICH (14).

Corin, a type II transmembrane serine protease mainly expressed in atrial and ventricular cardiomyocytes, plays a central role in cardiovascular regulation. It activates pro-atrial natriuretic peptide (pro-ANP) and pro-B-type natriuretic peptide (pro-BNP) by cleaving them into active ANP and BNP, which regulate blood pressure, natriuresis, diuresis, and cardioprotection (15-17). Beyond cardiovascular roles, corin is involved in embryonic development, particularly cardiac development, endometrial decidualization (preparing for pregnancy), osteogenic differentiation of bone marrow mesenchymal stem cells, adipose function, and thermoregulation (15, 18, 19). Notably, clinical studies have found significantly reduced soluble corin levels in patients with hemorrhagic stroke (20), and emerging evidence links soluble corin to hemorrhagic stroke risk factors, recurrence, and adverse outcomes (21, 22). These findings suggest that corin contribute to the pathophysiology of ICH; however, the underlying mechanisms remain unclear.

Overall, these results reveal a previously unrecognized corin–AMPK axis that regulates microglial homeostasis and mitochondrial resilience after ICH. Targeting this pathway could provide new therapeutic strategies to reduce secondary brain injury.

RESULTS

Corin overexpression improves ICH-induced neurological deficits in rats

Corin mRNA and protein levels in rat brain tissues showed a progressive decline at days 0, 1, 3, 7, and 14 after ICH induction, reaching their lowest levels at day 3 (Fig. 1A, B). At this time point, corin mRNA and protein levels were reduced to approximately 18.7% and 21.3% of baseline levels, respectively (P < 0.001) (Fig. 1A, B). These findings indicate that corin expression is substantially suppressed during ICH pathology. To investigate the functional role of corin, a corin-expressing lentivirus or control vector was administered via intracerebroventricular injection before ICH induction. After corin overexpression, corin mRNA and protein levels increased by 3.74 and 2.94 folds (P < 0.001) (Fig. 1C, D). Neurological function was assessed using the Neurological deficit score (NDS) and the MWM. Compared with control rats, ICH rats exhibited significant neurological and cognitive impairments, including increased NDS and escape latency, reduced time in the target quadrant, and longer swimming path length (Fig. 1E-H). Conversely, corin overexpression significantly improved neurological outcomes, demonstrated by a 34.9% reduction in NDS (P < 0.001), a 69.2% reduction in escape latency on day 4 (P < 0.001), a 2.1-fold increase in time spent in the target zone (P < 0.05), and a shorter swimming path length (Fig. 1E-H). These results indicate that corin overexpression effectively mitigates ICH-induced neurological deficits.

Fig. 1.

Effects of corin overexpression on ICH-induced neurological deficits in rats. Temporal changes in corin (A) mRNA and (B) protein levels in rat brain tissues post-ICH modeling (Days 0, 1, 3, 7, 14), assessed by qRT-PCR and Western blotting, respectively. Rats received intracerebroventricular injections of corin overexpression lentivirus or blank lentivirus 5 days prior to ICH modeling. Corin (C) mRNA and (D) protein levels in brain tissues were measured by qRT-PCR and Western blotting. Neurological deficits were assessed post-ICH by (E) neurological deficit score (NDS), (F) escape latency, (G) time spent in the target zone and (H) swimming paths in the MWM test. Data presented as mean ± SD (n = 3 or 5, independent experiments). Significant differences between groups were assessed using one-way ANOVA with Tukey’s post-hoc test. *P < 0.05, **P < 0.01, ***P < 0.001 vs. 0 d or control. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. ICH＋vector.

Corin overexpression attenuates microglial activation and inflammatory response after ICH

To investigate the relationship between corin and microglia in rats with ICH, we performed immunofluorescence staining to assess corin expression in microglial cells. Compared with control rats, ICH rats showed markedly enhanced microglial activation, evidenced by a 2.5-fold increase in Iba-1⁺ microglia and a 71.2% reduction in corin^＋Iba-1^＋ microglia (P < 0.001) (Fig. 2A-C). We further assessed microglial inflammatory responses in control rats, ICH rats receiving pre-lentiviral injection, and ICH rats with intracerebroventricular delivery of a corin-expressing lentivirus. Relative to controls, ICH rats exhibited a 4.3-fold increase in iNOS^＋ cells and a 67.7% decrease in Arg-1^＋ cells (P < 0.001) (Fig. 2D-F). In parallel, levels of proinflammatory cytokines were significantly elevated in ICH brain tissue, including TNF-α (4.0-fold), IL-1β (2.2-fold), and IL-6 (4.0-fold) (P < 0.001) (Fig. 2G), indicating pronounced microglial inflammatory activation. As shown in Fig. 2A-G, corin overexpression significantly corrected these pathological changes. It increased the corin^＋Iba-1^＋ microglia population by 2.8 folds (P < 0.01), reduced Iba-1^＋ and iNOS^＋ cell population by 53.1% and 71.4% (P < 0.001), and doubled the Arg-1^＋ cell population (P < 0.01). In addition, corin overexpression decreased the release of TNF-α, IL-1β, and IL-6 by 45.7%, 34.0%, and 39.1%, respectively (P < 0.001), demonstrating its inhibitory effect on microglial inflammatory polarization after ICH.

Fig. 2.

Effects of corin overexpression on microglial activation and inflammatory response after ICH. Rats received intracerebroventricular injections of corin overexpression or blank lentivirus 5 days pre-ICH. (A-C) Immunofluorescence staining measured corin and Iba-1 expression in brain tissues 3 days post-ICH. (D-F) Immunofluorescence staining measured iNOS and Arg-1 expression in brain tissues 3 days post-ICH. (G) Following identical lentiviral pretreatment, TNF-α, IL-1β, and IL-6 levels in brain tissues were quantified by ELISA 3 days post-ICH. Data presented as mean ± SD (n = 3 or 5, independent experiments). Significant differences between groups were assessed using one-way ANOVA with Tukey’s post-hoc test. Scale bar, 25 μm. ***P < 0.001 vs. control. ^##P < 0.01, ^###P < 0.001 vs. ICH＋vector.

Corin overexpression alleviates OGD/H-induced cell migration and inflammatory response in HAPI microglial cells

Corin mRNA and protein levels progressively decreased with increasing durations of OGD/H treatment (Fig. 3A-C), consistent with the findings in ICH rat models. To investigate the functional role of corin, HAPI microglial cells were transduced with a corin-expressing lentivirus. Compared with OGD/H model cells, corin overexpression resulted in a 1.2-fold increase in cell viability (P < 0.05) (Fig. 3D), a 1.8-fold increase in migration capacity (P < 0.01) (Fig. 3E), significant reductions in iNOS and Iba-1 expression by 60.8% and 48.9%, respectively (P < 0.01) (Fig. 3F, G). In addition, Arg-1 expression increased by 4.8 folds (P < 0.001) (Fig. 3G), indicating a shift toward a pro-repair microglial phenotype.

Fig. 3.

Effects of corin overexpression on OGD/H-induced cell migration and inflammatory response in HAPI microglia. (A) Corin mRNA and (B, C) protein levels in HAPI cells after OGD/H treatment (0, 3, 6, 12, 24 h). (D-G) Cells pre-transduced for 24 h with corin overexpression or blank lentivirus were subjected to 24 hrs OGD/H, followed by assessment of (D) cell viability, (E) migration capacity, (F) iNOS/Arg-1 mRNA levels and (G) corin/Iba-1 protein expression. Data presented as mean ± SD (n = 3). All experiments were repeated three times. Significant differences between groups were assessed using one-way ANOVA with Tukey’s post-hoc test. *P < 0.05, **P < 0.01, ***P < 0.001 vs. 0 h or control. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. OGD/H＋vector.

Corin overexpression boosts mitochondrial function in OGD/H-stimulated HAPI microglial cells

To determine whether mitochondrial restoration contributes to the protective effects of corin under OGD/H conditions, mitochondrial function was assessed following corin lentiviral transduction in HAPI microglia. Compared with OGD/H model cells, corin overexpression significantly increased the oxygen consumption rate (OCR) (Fig. 4A), elevated mitochondrial complex I-V activities by 1.6-2.9 folds (P < 0.05) (Fig. 4B-F), and enhanced ATP production by 1.9 folds (P < 0.001) (Fig. 4G). In addition, corin overexpression significantly increased the p-AMPK/AMPK ratio by 2.0 folds (P < 0.05) (Fig. 4H), suggesting activation of AMPK signaling and promotion of a pro-repair microglial state.

Fig. 4.

Effects of corin overexpression on OGD/H-induced mitochondrial dysfunction in HAPI microglia. Following 24 h pre-transduction with corin overexpression or blank lentivirus, cells underwent 24 h OGD/H exposure. Measurements included (A) OCR, (B-F) Mitochondrial complex activities (I, II, III, IV, V), (G) ATP generation, (H) p-AMPK/AMPK protein levels. Data presented as mean ± SD (n = 3). All experiments were repeated three times. Significant differences between groups were assessed using one-way ANOVA with Tukey’s post-hoc test. ***P < 0.001 vs. control. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. OGD/H＋vector.

Corin overexpression inhibits OGD/H-induced cell migration, inflammatory response and mitochondrial dysfunction via the AMPK signaling pathway

To determine whether AMPK activation mediates the protective effects of corin against OGD/H-induced microglial injury, corin-overexpressing HAPI cells were treated with Compound C, an AMPK inhibitor that suppresses AMPK phosphorylation and kinase activity under pathological conditions. Compared with corin-overexpressing cells alone, Compound C treatment resulted in a 12.9% reduction in cell viability (P < 0.05; Fig. 5A) and a 34.0% decrease in cell migration (P < 0.01) (Fig. 5B). It also caused a 23.3-fold increase in iNOS expression and a 86.3% decrease in Arg-1 expression (P < 0.001) (Fig. 5C), indicating reversal of the corin-induced pro-repair microglial phenotype. Furthermore, Compound C led to a 1.9-fold increase in Iba-1 expression and a 65.4% decrease in the p-AMPK/AMPK ratio (P < 0.001) (Fig. 5D). Mitochondrial function was also markedly impaired, as evidenced by reduced OCR (Fig. 5E), a 36.4%-48.5% decrease in mitochondrial complex I-V activities (P < 0.01) (Fig. 5F-J), and a 45.9% reduction in ATP production (P < 0.001) (Fig. 5K).

Fig. 5.

Overexpression of corin mitigates OGD/H-induced cell migration impairment, inflammatory response, and mitochondrial dysfunction in HAPI microglia via the AMPK pathway. Cells were pre-transduced with corin-overexpressing or control lentivirus for 24 h, followed by OGD/H and treatment with 10-μM Compound C for 24 h. Subsequent assessments included: (A) cell viability, (B) migration, (C) iNOS and Arg-1 mRNA levels, (D) Iba-1, p-AMPK, and AMPK protein levels, (E) OCR, (F-J) activities of mitochondrial complexes I-V, and (K) ATP generation. Data presented as mean ± SD (n = 3). All experiments were repeated three times. Significant differences between groups were assessed using one-way ANOVA with Tukey’s post-hoc test. *P < 0.05, **P < 0.01, ***P < 0.001 vs. OGD/H＋vector＋vehicle. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. OGD/H＋Corin+vehicle.

DISCUSSION

This study identifies a previously unrecognized neuroprotective role of corin in ICH. We demonstrate for the first time that endogenous corin expression is significantly downregulated in perihematomal brain tissue following ICH, reaching its lowest level at 72 h post-injury—a critical period for secondary brain damage. Importantly, targeted corin overexpression provided significant protection against ICH-induced pathology through multiple mechanisms, including improvement of neurological function and suppression of neuroinflammation.

Our findings establish corin as a key neuroprotective factor in ICH-induced brain injury. Beyond its classical cardiovascular functions, accumulating evidence indicates that corin exerts pleiotropic effects, such as regulating osteogenic differentiation (15, 23), modulating chemotactic signaling and inflammation (24), and participating in adipose thermoregulation (19), through both ANP-dependent and ANP-independent pathways. Considering its involvement in shared cardiovascular mechanisms, soluble corin has been associated with cardiovascular disorders—including heart failure, hypertension, and stroke—and proposed as a diagnostic biomarker (21, 25, 26), although the relationship between circulating soluble corin and membrane-bound corin remains unclear. These observations prompted us to explore the role of corin in the brain during ICH pathology. Our data demonstrate a clear reduction of cerebral corin expression after ICH, while intracerebroventricular administration of corin-expressing lentivirus significantly alleviated neurological deficits. Collectively, these results highlight the therapeutic potential of corin and provide new insight into its role in the pathophysiology of ICH, offering a promising direction for future therapeutic development.

Our findings demonstrate that corin provides broad neuroprotection across multiple pathological features of ICH by inhibiting microglial activation and dampening inflammatory responses in both injured brain tissue (in vivo ICH model) and activated microglia (in vitro OGD/H model). As central orchestrators of CNS immune homeostasis (27), microglia dynamically shift between pro- and anti-inflammatory phenotypes to influence disease progression. Importantly, microglia-driven neuroinflammation is a central mechanism underlying secondary injury after ICH (28), making its modulation crucial for improving neurological outcomes (27, 28). Our data identify corin as a pivotal regulator of microglial-mediated neuroinflammation, highlighting its potential as a therapeutic target to mitigate post-ICH brain damage through control of inflammatory responses.

This study further reveals that activation of AMPK signaling and restoration of mitochondrial function are essential mechanisms underlying the neuroprotective effects of corin. Mitochondrial dysfunction and AMPK inactivation are well-established drivers of microglial phenotypic polarization (29). Following ICH, perihematomal ischemia/hypoxia and mitochondrial reactive oxygen species generated during erythrocyte lysis contribute to profound mitochondrial impairment (30, 31). AMPK, a critical cellular energy sensor, exhibits reduced activity after ICH and is associated with aggravated mitochondrial damage and immune dysregulation (32, 33). Both mitochondrial integrity and AMPK signaling are widely recognized as essential components of post-ICH repair processes across cell types, including microglia (13). In this study, corin overexpression restored mitochondrial function and enhanced AMPK activation in OGD/H-stimulated microglia. The critical role of AMPK was further confirmed by pharmacological inhibition experiments: treatment with Compound C abolished the protective effects of corin, including its suppression of microglial migration, inflammatory responses, and mitochondrial dysfunction. Together, these findings define a novel corin–AMPK signaling axis that regulates microglial activation, neuroinflammation, mitochondrial homeostasis, and ultimately neuronal survival and functional recovery. This pathway represents a promising therapeutic target for reducing secondary brain injury following intracerebral hemorrhage.

This work has several limitations. Although our results identify corin–AMPK signaling as a critical regulator of microglial function, it remains to be determined whether this pathway exerts similar effects in other neural cell types, astrocytes and neurons. Prior studies have demonstrated that corin promotes gastric cancer progression through activation of the ERK1/2 pathway (34) and alleviates kidney fibrosis and cardiomyocyte injury via Wnt/β-Catenin, PI3K/AKT, and NF-κB signaling pathways (35, 36). Therefore, further investigation into additional corin-mediated signaling cascades is necessary to fully elucidate its neuroprotective mechanisms. Considering that ERK and AMPK signaling are both involved in regulating mitochondrial morphology and dynamics (37), it will be important to determine whether ERK signaling directly or indirectly contributes to the protective effects of corin in ICH. Moreover, the in vivo delivery efficiency and long-term safety of corin-based gene therapy approaches require further optimization before clinical translation. Finally, identifying the upstream mechanisms responsible for corin downregulation following ICH may uncover new therapeutic targets to prevent or reverse secondary brain injury.

CONCLUSIONS

This study advances our understanding of ICH pathophysiology by identifying corin and its downstream AMPK signaling as key regulators of neuroinflammation and neuronal injury, thereby revealing a promising therapeutic axis for future intervention strategies.

MATERIALS AND METHODS

Animal and experimental groups

Adult male Sprague–Dawley rats (280-300 g) were obtained from Hunan SJA Laboratory Animal Co., Ltd. (Hunan, China). After a 7-day acclimatization period in a controlled environment (22°C ± 1°C, 12-h light/dark cycle), rats were assigned to the following groups (n = 5 per group unless otherwise specified): sham, ICH-1 d, ICH-3 d, ICH-7 d, ICH-14 d, ICH^＋vector (ICH modeling + blank lentivirus), and ICH^＋Corin (ICH modeling + Corin overexpression lentivirus). All experimental protocols received approval from the Medical Ethics Committee of Huzhou Central Hospital (approval number 202209012-01) and adhered to institutional animal care and use guidelines.

ICH modeling in vivo

ICH was induced in rats using a stereotactic autologous blood injection method. Briefly, rats were anesthetized with 2% isoflurane, and 50 μl of blood was collected from the femoral artery. Rats were then secured in a stereotactic apparatus, and 100 μl of the collected autologous blood was slowly injected into the right basal ganglia (coordinates: 0.2 mm anterior, 3 mm lateral to bregma; depth 5.8 mm) at a rate of 25 μl/min using a microinjection pump. After injection, the needle was help in place for 10 min to prevent backflow, then withdrawn gradually at 1 mm/min. The puncture site was sealed with bone wax, and the scalp incision was sutured. Control rats received an equivalent volume of sterile saline at the same coordinates under identical conditions. Animals were euthanized at 0, 1, 3, 7, and 14 days following ICH induction, and brain tissues were collected for quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analyses.

Lentiviral injection to ICH rats

Five days before ICH induction, either corin overexpression lentivirus or a blank lentivirus (titer: 1 × 10⁹ TU/ml) was stereotactically injected into the lateral ventricle (coordinates: 1 mm anterior, 2 mm lateral to bregma; depth 3.5 mm). The viral solution was infused at a rate of 0.5 μl/min over 10 min. Rats were euthanized three days post-ICH induction, and brain tissues were harvested for qRT-PCR and Western blot analyses.

Neurological deficit score (NDS)

Neurological function was evaluated at 3 days post-ICH using a 28-point scale encompassing seven parameters: whisker response, compulsory circling, front limb symmetry, circling behavior, climbing ability, gait, and body symmetry. Each parameter was scored from 0 to 4, resulting in a total score range of 0-28 (0 = normal function; 28 = maximal deficit), following established protocols.

Morris water maze (MWM) test

Spatial learning and memory were assessed 7 days after ICH using the MWM according to established protocols (38). The test consisted of four daily training trials over five consecutive days in the afternoon. Escape latency was recorded to evaluate spatial learning, while the time spent in the target quadrant was used to assess spatial memory among the groups.

Immunofluorescence

Rat brains were subjected to fixation in paraformaldehyde (4%) for 48 h, dehydration in gradient ethanol, and vitrifiability in xylene. Thereafter, brains were embedded in paraffin and sectioned (20 μm thickness). For immunofluorescence staining, sections were incubated overnight with corin (Abcam; ab255812), Iba-1 (Proteintech; 81728-1-RR), iNOS (Servicebio; GB11119), and Arg-1 (Servicebio; GB11285) antibodies at 4°C, followed by Alexa Fluor 488-labeled Goat Anti-rabbit IgG (H＋L) (Beyotime; A0423), Alexa Fluor 555-labeled Donkey Anti-rabbit IgG (H＋L) (Beyotime; A0453), and Alexa Fluor 555-labeled Donkey Anti-mouse IgG (H＋L) (Beyotime; A0460) antibodies at 25°C. Nuclei were stained by DAPI (Beyotime; C1002). Immunofluorescence confocal microscopy was used to examine stained cells.

Enzyme‑linked immunosorbent assay (ELISA)

Brain tissue cytokine concentrations (TNF-α, EK382/3; IL-1β, EK301B/3; IL-6, EK306/3) were quantified using ELISA according to manufacturer protocols (Multi Sciences, Hangzhou, China).

Cell culture

Rat HAPI microglial cells (Procell, Wuhan, China) were used to establish an in vitro ICH model through oxygen–glucose deprivation plus hemin (OGD/H) as described (39). Briefly, cells were pre-transduced for 24 h with corin overexpression lentivirus or blank lentivirus (Genepharma, Shanghai) and subsequently subjected to OGD/H for 24 h through culturing in glucose/serum-free DMEM (Gibco, USA) under 95% N₂/5% CO₂ (10 mins), treated with 10-μM hemin (Sigma-Aldrich, Germany) in the same atmosphere (120 mins). Cells were then returned to normal culture conditions. For AMPK inhibition, lentivirus-transduced cells underwent OGD/H co-treated with 10-μM Compound C (Selleck, Shanghai) for 24 h.

Construction of lentiviral cell lines

Corin overexpression lentivirus was generated using the pLVX-Puro system. HEK-293T cells were co-transfected with either blank pLVX-Puro or corin overexpression lentiviral vectors plus pMD2G/psPAX2 packaging plasmids using Lipofectamine 2000 (Thermo-Fisher). Lentiviral supernatant was harvested at 48 h post-transfection for subsequent transduction of HAPI microglial cells.

Cell Counting Kit-8 (CCK-8) assay

Following overnight culture at 37°C, HAPI microglial cells were treated with 10-μl CCK-8 reagent (Beyotime, C0039) for 1 h. Cell viability was determined by measuring 450-nm absorbance using a microplate reader (BioTek Epoch2, USA).

Transwell assay

HAPI microglial cells (3 × 10⁴ cells/well) were seeded in 6-well plates and cultured overnight at 37°C. For migration assays, 2 × 10⁴ cells in serum-free DMEM were loaded into Transwell chambers (Costar), with complete medium in the lower compartment. After 24 h incubation, cells were fixed with 4% paraformaldehyde (1 ml, 10 mins) and stained with 0.5% crystal violet (1 ml, 30 mins). Migrated cells were imaged using a microscope.

Extracellular flux analysis

The OCR was measured by extracellular flux analysis. Cells (1 × 10⁴/well) were seeded in XF-24 plates and cultured for 24 h (37°C, 5% CO₂). Before assay, cells were equilibrated in a CO₂-free incubator with XF Base Medium. Mitochondrial stress was assessed using the Seahorse XF Kit (Agilent, 103015-100) with sequential inhibitor addition.

Mitochondrial complex activity and ATP determination

Mitochondrial complex I-V activities (BC0515, BC3230, BC3240, BC0940, BC1440; Solarbio) and ATP generation (A095-1-1; Nanjing Jiancheng) were measured using commercial kits per manufacturer’s protocols.

qRT-PCR

Total RNA was extracted using TRIzol (Thermo-Fisher), reverse-transcribed with PrimeScript cDNA Synthesis Kit (Takara), and subjected to qRT-PCR with SYBR Green Master Mix (Thermo-Fisher). Gene expression was normalized to β-actin and quantified via the 2^−∆∆CT method. Primer sequences included corin-F: 5ʹ-CAGAGTCCCTCCAACCACCG-3ʹ; corin-R: 5ʹ-CCTGGAACTTGGACCCGGAG-3ʹ; iNOS-F: 5ʹ-AACAACGTGGAGAAAACCCC-3ʹ; iNOS-R: 5ʹ-GGGTCGATGGAGTCACATGC-3ʹ; Arg-1-F: 5ʹ-CCAAGCCAAAGCCCATAGAGA-3ʹ; Arg-1-R: 5ʹ-CAGGCCAGCTTTCCTTAATGC-3ʹ; β-actin-F 5ʹ-CCCTAAGGCCAACCGTGAA-3ʹ, β-actin-R 5ʹ-ATGCCAGTGGTACGACCAGA-3ʹ.

Western blotting

Whole-cell lysates were prepared using RIPA buffer (Beyotime) supplemented with protease inhibitors. Equal amounts of protein (20 μg) were separated by SDS-PAGE and transferred onto PVDF membranes (Millipore). Membranes were incubated overnight at 4°C with primary antibodies targeting Corin (Proteintech, 83566-1-RR), Iba-1 (Proteintech, 81728-1-RR), phosphorylated AMPK (p-AMPK; Abclonal, AP1002), AMPK (Proteintech, 10929-2-AP), and β-actin (Proteintech, 66009-1-Ig). Following three washes with TBST, membranes were incubated for 1 hour at room temperature with HRP-conjugated secondary antibodies (ZSGB-BIO, ZB-2301/2305). Protein bands were visualized using an ECL detection kit (Pierce) and quantified with Image-Pro Plus 6.0 software, normalized to β-actin levels.

Statistical analyses

Data are expressed as mean ± SD and analyzed using GraphPad Prism version 8.4.2 (GraphPad Software, Inc., USA). Comparisons among groups were performed using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test. A P-value < 0.05 was considered statistically significant.