Neprilysin Inhibitor and Angiotensin Receptor Blocker Attenuate Ischemic Brain Injury: Unveiling the Kinin-Mediated Pathway

Abstract

Kinin peptides play a key role in regulating blood vessel tone, renal function, and protection against ischemia-reperfusion injury. Neprilysin is one of the primary enzymes responsible for degrading several vasoactive peptides including bradykinin. Concurrent inhibition of neprilysin and angiotensin II receptors leads to elevated bradykinin levels by reducing its breakdown, potentially enhancing its interaction with bradykinin receptor complexes. It has therefore been hypothesized that increased bradykinin generation may contribute to the neuroprotective effect of neprilysin inhibitors and angiotensin receptor blockers against global cerebral ischemia-reperfusion injury (GCI/R). GCI/R was induced in mice by transient occlusion of bilateral common carotid arteries. A combination of Sacubitril/Valsartan (30/50 mg/kg and 60/100 mg/kg, PO) was administered for 7 days before subjecting to ischemia. Behavioral changes were assessed using the beam walk test for motor coordination and passive avoidance and Y-maze tests for spatial memory, which were further supported by biochemical and histopathological assessment. Our results demonstrated that prophylactic treatment with Sacubitril/Valsartan led to a dose-dependent improvement in neurological deficits, a reduction in oxidative stress and inflammation, and an increased number of intact neurons in the hippocampus and cortex of GCI/R mice. Interestingly, immunohistochemical analysis revealed upregulation of bradykinin B2 receptors accompanied by changes in eNOS and NFκB expression. However, coadministration of the bradykinin B2 receptor antagonist, Icatibant, attenuated the neuroprotective effects of Sacubitril/Valsartan. Our findings suggest that the neuroprotective effects of Sacubitril/Valsartan may be mediated by the endogenous accumulation of vasodilatory bradykinin. Thus, concurrent inhibition of neprilysin and angiotensin II receptors represents a promising therapeutic target for stroke management.

Keywords

Bradykinin Cerebral ischemia-reperfusion injury Neprilysin Neuroprotection Sacubitril Valsartan

INTRODUCTION

Most patients who survive cardiac arrest experience residual global cerebral ischemia/reperfusion injury (GCI/R) and associated neurological impairments such as motor dysfunction, aphasia, cognitive deficits and even coma.^1,2 Such neurological injuries, occurring because of reperfusion following cardiopulmonary resuscitation (CPR), result in poor quality of life and impose an economic and psychological burden on both families and communities.³ The pathogenesis of GCI/R injury post-CA is complex and involves excitotoxicity, acidosis, ionic imbalance, oxidative stress, inflammation, apoptosis, and ultimately irreversible neuronal damage.⁴ Despite various attempts to target these pathophysiological processes with neuroprotective strategies, significant progress in improving patient outcomes following cerebral ischemia remains elusive, aside from the advancements made with thrombectomy.^5,6 Therefore, there is an urgent need to develop more effective neuroprotective strategies, to increase tissue salvage and enhance functional recovery post-GCI/R injury. Among experimental approaches, the bilateral common carotid artery occlusion (BCCAO) rodent model is particularly effective in simulating chronic brain hypoperfusion observed in humans, making it an ideal method for inducing global cerebral ischemia in research settings.^7,8

Bradykinin (BK), the most bioactive kinin in mammals, is synthesized in the brain via the kallikrein-kinin pathway. BK has important physiological actions related to regulating blood vessel tone and renal functions, and protection from ischemia-reperfusion injury.^9,10 Of the two receptor types bradykinin B1 and B2, B2 receptors are predominantly expressed and play a central role in mediating the key effects of BK.^11,12 Neprilysin, known as neutral endopeptidase (NEP), degrades various bioactive peptides, including natriuretic peptides (NPs), and neuropeptides such as Substance P, enkephalins and bradykinin. Recently, Neprilysin inhibitors have gained prominence in the treatment of cardiovascular diseases due to their ability to increase plasma concentration of these essential peptides; since they have natriuretic and vasodilating effects, also inhibit the renin–angiotensin–aldosterone system and exert antihypertrophic and antiproliferative effects.^13,14

Sacubitril (SAC) is a prodrug that is metabolized into LBQ657, a well-tolerated neprilysin inhibitor. The combined inhibition of neprilysin and angiotensin II receptors has been proven to offer clinical protection in a range of cardiovascular diseases, including hypertension and heart failure.¹⁵ Previous studies reported that sacubitril/valsartan (SAC/VAL) has additional protective roles; it could reduce Paraquat-induced lung injury, Doxorubicin-induced cardiotoxicity, and Diabetes-induced cardiomyopathy and improve renal function in chronic kidney diseases.^16,17 The conflicting evidence in the literature has raised the concerns about long-term neurocognitive safety of angiotensin receptor-neprilysin inhibitors (ARNIs).¹⁸ However, in a cross-sectional study of patients with heart failure, SAC/VAL improved cerebral perfusion and reduced proinflammatory cytokines contributing to lower rates of neurocognitive disorders.¹⁹ Bai et al. demonstrated that LCZ696 pretreatment prevented ischemic brain damage after stroke by increasing cerebral blood flow.²⁰ Although the literature suggests that elevated levels of natriuretic peptides and bradykinin may contribute to the therapeutic benefits of neprilysin inhibitors, their precise role in neuroprotection during cerebral ischemia remains elusive.

Therefore, this study is designed to investigate the potential neuroprotective effects of SAC/VAL on global cerebral ischemia-reperfusion injury induced by BCCAO in mice. We hypothesize that SAC/VAL may attenuate neuronal injury by modulating the Bradykinin B2 receptor/eNOS signaling pathway, thereby supporting its therapeutic application in promoting functional recovery post-GCI/R injury.

MATERIALS AND METHODS

Drugs and Chemicals

Sacubitril and Icatibant (ICT) acetate were purchased from Sigma-Aldrich, USA. Valsartan was supplied by Yarrow Chemicals Pvt. Ltd., India. The drug solutions (SAC and VAL) were freshly prepared by suspending 0.5% carboxymethyl cellulose (CMC).²¹ All other chemicals used in the study were of analytical reagent (AR) grade.

Experimental Animals and Treatment Groups

Animal experimentation was performed following the guidelines set by the Committee for the Control and Supervision of Experimentation on Animals (CCSEA) and approved by the Institutional Animal Ethical Committee of Raj Kumar Goel Institute of Technology (Pharmacy) Ghaziabad, India (Approval no: RKGIT/IAEC/2023/09). Male Swiss albino mice (18–30 g), obtained from the institutional animal facility, were provided food and water ad libitum and housed under controlled conditions: 22–24°C and a 12 h light/dark cycle. The mice were randomly divided into the following five groups (n = 8): Group I: Sham received vehicle of 0.5% CMC, Group II: I/R model group, where bilateral common carotid arteries were occluded to induce global cerebral ischemia and reperfusion injury. Group III and Group IV were treated with a combination of SAC/VAL at doses of 30/50 mg/kg and 60/100 mg/kg, respectively, administered orally (PO) for 7 days before subjecting to global cerebral ischemia. Group V received SAC/VAL (60/100 mg/kg, PO)²¹ along with ICT (1 mg/kg, i.v.)²² administered 30 min before the induction of ischemia. Behavioral parameters were assessed 24 h after reperfusion.

Global Cerebral Ischemia and Reperfusion Model

The bilateral common carotid artery occlusion method was used to induce global cerebral ischemia-reperfusion in mice.²³ The mice were anesthetized by intraperitoneal injection of ketamine (80 mg/kg) and xylazine (5 mg/kg) solution. The targeted area was shaved and sterilized, following which a ventral midline incision was made in the cervical region, and the bilateral carotid arteries were carefully separated from the vagus nerve. A suture (Trulene 2-0) was passed below each carotid artery, and global cerebral ischemia was induced by ligating the sutures, which were subsequently removed 17 min later. The incision was then closed and betadine solution was applied to prevent infection. In the sham group, no occlusive procedure was performed. Following surgery, each mouse was given anti-infective and routine care.

Behavioural Tests

Beam walk test

This behavioral task was used to assess motor coordination and balance in mice postischemia. Each animal was individually placed on a metallic bar 120 cm long and 2 cm wide, placed 70 cm above the ground, and allowed to walk across the beam. The motor performance was evaluated by measuring the time to traverse the beam and the number of hindlimb foot slips. Three trials per mouse were performed and the average was used for analysis.²⁴

Y-maze test

This model is extensively used to assess the spatial working memory of rodents by evaluating their general exploratory behavior. The apparatus consists of a wooden Y maze with three distinct arms (A, B, and C) arranged at an angle of 120°, with dimensions measuring 50 cm × 10 cm × 18 cm. The protocol consisted of two sessions: an initial training session succeeded by a subsequent test session conducted the following day, after 24 h of reperfusion. Each mouse was initially placed at the center of the maze juncture and allowed to freely explore the maze for 10 min. The total number of arm entries and alternations were recorded. An animal entering an arm with 85% of its body was considered an entry and successive entries in three distinct arms of Y-maze were counted as one alternation.²⁵ To minimize the influence of odor cues on the animal’s behavior, the Y-maze was cleaned with 75% alcohol between each trial. The percentage of spontaneous alternations (SA%) was calculated by the following formula:

Spontaneous alternation (%) = \frac{no . of spontaneous alternations}{Total number of arm entries - 2} \times 100

Passive avoidance test

The passive avoidance test is used to assess avoidance memory retention in mice. The apparatus comprised two compartments: an illuminated box and a dark box, separated by a guillotine door. On the first day of testing, before surgery, the mice were placed in the apparatus for 5 min to habituate, with the door open and animal-free to explore both compartments. Thirty minutes after habituation, during the acquisition trial, the mice were placed back into the illuminated box with the door raised. A mild electric shock (0.75 mA, 3 s) was delivered when the mouse entered the dark box. The retention test was conducted 24 h later using the same procedure as the acquisition trial, but without giving an electric shock. In both the acquisition and retention trials, the transfer latency (i.e., time taken for the mouse to enter the dark box) was recorded in seconds with a cut-off time of 300 s.²⁶

Biochemical Estimation

At the end of 24 h reperfusion, the mice were euthanized to isolate the brain tissue. The brain tissue was rinsed with ice-cold isotonic saline and then homogenized with ice-cold 0.1 M phosphate buffer (pH 7.4) in ratio of tissue weight (mg): buffer volume µL = 1:9. The homogenate was centrifuged at 10,000 g for 15 min at 4°C and aliquots of supernatant were used for biochemical analysis.

Estimation of MDA

The concentration of malondialdehyde (MDA) in the tissue supernatant was estimated using thiobarbituric acid reactive substances assay.²⁷ The OD was measured spectrophotometrically at 532 nm and expressed as nanomoles of MDA formed per mg of protein. The protein amount of the brain sample was calculated by Lowry’s method.²⁸

Estimation of GSH and Catalase

The total glutathione (GSH) and catalase activity in the supernatant were estimated as per the manufacturer’s instructions using commercially available kits (Caymen Chemicals, USA).

Pro-Inflammatory Cytokines

The estimation of tumor necrosis factor- alpha (TNF-α) and interleukin-6 (IL-6) levels in the brain was done by their respective mouse Enzyme-Linked Immunosorbent Assay (ELISA) kits (Abbkine, China). All the procedures were performed according to the manufacturer’s instructions.

Hematoxylin-Eosin Staining Assay

Hematoxylin and Eosin staining was performed in the hippocampal and cortex region to evaluate the histopathological changes following GCI/R injury in mice. After euthanizing the mice, the brain tissues were quickly isolated, fixed in formalin, and embedded in paraffin. Coronal sections of 4 µm were stained with hematoxylin to visualize the nuclei and eosin to stain the cytoplasm and extracellular matrix and observed under the microscope under high magnification value (x400). The key pathophysiological changes such as neuronal loss, nuclear pyknosis, vacuolation, and disrupted cell organization were identified.²⁹ The number of intact and damaged neurons was counted using Image J software.

Immunohistochemistry

Immunohistochemistry was performed to detect the expression levels of the Bradykinin B2 receptor and eNOS proteins. Coronal sections with a thickness of 4 µm at the cortical level were dewaxed and subsequently processed for immunohistochemical staining. After eliminating the endogenous peroxidase activity, the slides were thoroughly rinsed with phosphate-buffered saline multiple times and preincubated in 1% bovine serum albumin for 45 min at room temperature; thereafter, the slides were incubated overnight with primary antibodies, Rabbit BDKRB2 mouse antibody (1:100, sigma) or mouse ant-eNOS antibody (1:200, sigma) or NFκB monoclonal mouse (1:300, sigma). Subsequently, the sections were treated with the secondary antibody, biotinylated goat anti-rabbit (1:1000).³⁰ The slides were then subjected to treatment with 3,4-diaminobenzidine and examined under a light microscope. Image J software was employed to quantitatively analyze the immune-positive cells.

Statistical Analysis

All data are expressed as mean ± standard error of the mean (SEM) and were statistically analyzed using GraphPad Prism 10.2.3. The one-way analysis of variance (ANOVA), followed by post hoc Tukey’s test, was used to compare the differences between the groups in all pretreatment groups. The p < 0.05 were considered statistically significant.

RESULTS

SAC/VAL Ameliorated Cognitive and Neuromuscular Impairment Induced by GCI/R Injury

Figure 1 illustrates the effect of SAC/VAL on memory impairment in GCI/R mice, assessed using the Y-maze and passive avoidance test paradigms. One-way ANOVA revealed a significant (p = 0.002) difference in the total number of arm entries among the groups in the Y-maze paradigm ( Figure 1A ). The I/R group showed a notable (p < 0.05) reduction in the total number of arm entries compared with the sham group, indicating reduced exploratory behavior following GCI/R injury. Treatment with SAC/VAL at both doses, 30/50 mg/kg and 60/100 mg/kg, alleviated the ischemia-induced decline in curiosity, with the 60/100 mg/kg dose producing a more pronounced (p < 0.05) improvement in exploratory activity than the 30/50 mg/kg dose.

Fig. 1.

Effect of sacubitril/valsartan (SAC/VAL) pretreatment on global cerebral ischemia-reperfusion induced changes in neurobehavioral deficits in mice; cognitive impairment is represented as total number of arm entries (A) and percentage of spontaneous alterations (B) in Y-maze test, Step-through latency (sec) in passive avoidance test (C); motor coordination is represented as number of foot slips (D) and time taken to cross the beam (E) in beam walk test. One-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons was used; values are expressed as mean ± SEM (n = 6), ^#p < 0.05 versus sham, **p < 0.01, *p < 0.05 versus I/R (ischemic-reperfusion) group, ^$p < 0.05 versus SAC/VAL (60/100 mg/kg) group. ANOVA, analysis of variance.

Spontaneous alteration behavior in the Y-maze test, which assesses spatial working memory, was significantly (p < 0.05) reduced in the I/R group compared to the sham group ( Figure 1B ). Administration of SAC/VAL at 30/50 mg/kg and 60/100 mg/kg doses significantly restored spontaneous alteration behavior, suggesting an improvement in spatial working memory. Furthermore, co-administration of ICT, a bradykinin B2 receptor antagonist, with SAC/VAL (60/100 mg/kg) significantly (p < 0.05) reduced the total number of arm entries and spontaneous alterations compared with the SAC/VAL 60/100 mg/kg group, suggesting a reversal of SAC/VAL’s beneficial effects on spatial working memory.

In the passive avoidance test, step-through latency (STL) was observed during both the acquisition and retention tests ( Figure 1C ). No significant differences were observed among the groups during the acquisition phase. The retention test, conducted 24 h later, revealed that I/R injury significantly (p < 0.05) reduced the STL compared with the sham group, indicating impaired memory retention. Prophylactic treatment with SAC/VAL at 60/100 mg/kg doses significantly (p < 0.05) increased the STL during the retention phase relative to the I/R group, suggesting improved memory performance. However, the presence of ICT with SAC/VAL (60/100 mg/kg) significantly diminished this effect, reducing STL and thereby reversing SAC/VAL’s beneficial effects on cognition.

In the narrow beam walk test, mice in the I/R group showed a significant increase in both the number of foot slips ( Figure 1D ) and time taken to traverse the beam ( Figure 1E ), indicating reduced muscle coordination compared to the sham group (p < 0.05). Pretreatment with SAC/VAL at both doses, 30/50 mg/kg and 60/100 mg/kg, significantly mitigated these deficits caused by I/R injury. However, co-administration of ICT with SAC/VAL (60/100 mg/kg) significantly (p < 0.05) negated the beneficial effects of SAC/VAL on motor impairment.

SAC/VAL Ameliorated the Oxidative Stress and Neuroinflammation Induced by GCI/R Injury

A significant rise in the MDA levels was observed in the I/R group compared to the sham group. Pretreatment with SAC/VAL at both 30/50 mg/kg and 60/100 mg/kg doses markedly reduced the elevated MDA levels in ischemic brain homogenate (p < 0.05). Conversely, I/R injury led to a significant decline in antioxidant levels, GSH and catalase in brain tissue, while SAC/VAL pretreatment at 30/50 mg/kg and 60/100 mg/kg doses effectively restored their levels (p < 0.05). However, co-treatment of ICT and SAC/VAL (60/100 mg/kg) significantly (p < 0.05) increased the MDA and decreased the GSH and catalase levels in the brain tissue, thereby reversing the beneficial effect of SAC/VAL treatment ( Figure 2 ).

Fig. 2.

Effect of sacubitril/valsartan on the GCI/R induced changes in cerebral oxidative stress markers: MDA (A), GSH (B) and Catalase (C). One-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons was used; values are expressed as mean ± SEM (n = 5), ^#p < 0.05 versus sham, **p < 0.01, *p < 0.05 versus I/R group, ^$p < 0.05 versus SAC/VAL (60/100 mg/kg) group. GCI/R, global cerebral ischemia-reperfusion injury; GSH, glutathione; MDA, malondialdehyde; SEM, standard error of the mean.

GCI/R injury initiated the neuroinflammatory response in mice, as indicated by higher TNF-α and IL-6 compared with the sham group (p < 0.05) ( Figure 3 ). However, SAC/VAL pretreatment (30/50 mg/kg and 60/100 mg/kg) significantly restored the levels of the proinflammatory cytokine compared with the I/R group. NFκB, the central regulator of the inflammatory response, mediates the transcription induction of these proinflammatory mediators. In the present study, ICH analysis revealed that GCI/R induced a significant increase in nuclear NFκB accumulation, which was markedly (p < 0.05) downregulated in SAC/VAL treatment groups relative to the I/R group ( Figure 3A–E ). However, cotreatment with ICT negated the protective effects of SAC/VAL on inflammation. These results suggest that SAC/VAL exhibits antioxidant and anti-inflammatory activity in GCI/R mice.

Fig. 3.

Effect of SAC/VAL on the neuroinflammatory markers in GCI/R mice; photomicrographs of IHC analysis (n = 3) showed upregulation of nuclear NFκB expression in I/R group (B) (indicated by red arrows) compared with the sham (A) (normal cells are depicted by green arrows); SAC/VAL administrated groups (C, D) successfully demonstrated the lower expression of NFκB compared with the I/R group, however, coadministration of Icatibant and SAC/VAL (60/100 mg/kg) (E) showed increased NFκB expression compared with SAC/VAL group, indicating reversal of its anti-inflammatory effect in the presence of Icatibant. Graphs representing the levels of TNF-α (F), IL-6 (G) (n = 5) and quantitative analysis of NFκB expression (H) in brain tissue. One-way ANOVA followed by Tukey’s post-hoc test for multiple comparisons was used; values are expressed as mean ± SEM, ^#p < 0.05 versus sham, ***p < 0.001, **p < 0.01, *p < 0.05 versus I/R group, ^$p < 0.05 versus SAC/VAL (60/100 mg/kg) group.

SAC/VAL Pretreatment Mitigated GCI/R-Induced Histopathological Alterations in Brain Tissue

H and E staining of the brain cortex and hippocampus region (400x, scale bar: 20 µm) showed that neurons in the sham group were intact, orderly arranged with clear round nuclei, and had no vacuoles, indicating structurally and functionally healthy neurons. The GCI/R group showed disorderly arranged cells with increased intercellular spaces, nuclear pyknosis, vacuolization, and many necrotic cells in the cortex and hippocampus, while SAC/VAL pretreatment at both doses (30/50 mg/kg and 60/100 mg/kg) reduced such morphological damage in the ischemic brain ( Figure 4A ). The quantitative analysis further supported these findings, indicating that the number of surviving neurons in the GCI/R group was significantly decreased compared with the sham group. Conversely, SAC/VAL pretreatment increased the number of intact neurons compared with the GCI/R group in a dose-dependent manner. However, co-treatment with ICT reduced the number of surviving neurons in the ICT + SAC/VAL group compared to the SAC/Val (60/100 mg/kg) group ( Figure 4B ).

Fig. 4.

Representative Hematoxylin and Eosin staining of the cortex and hippocampus are shown in each group (A) (n = 3) (400x, scale bar: 20 µm). % surviving neurons are quantitatively expressed as mean ± SEM (B). ^###p < 0.001 versus sham; **p < 0.01, *p < 0.05 versus I/R group; ^$p < 0.05 versus SAC/VAL (60/100 mg/kg) group.

SAC/VAL Pretreatment Activated the B2 Receptors and eNOS Expression in the Mice Brain Subjected to GCI/R Injury

Figure 5A and B shows the immunohistochemical staining of B2 receptors and eNOS-positive cells in the cortex of mice subjected to I/R injury, respectively. The results demonstrated that SAC/VAL treatment at both doses (30/50 mg/kg and 60/100 mg/kg) significantly increased the B2 receptors and eNOS positive ratio in GCI/R mice when compared with the I/R group. However, the presence of ICT significantly reduced the number of B2 receptors and eNOS-positive cells when compared to the SAC/VAL (60/100 mg/kg) group, indicating that endogenously produced bradykinin is involved in the neuroprotective actions of SAC/VAL. No statistically significant difference was observed between the sham and the I/R groups.

Fig. 5.

Effect of SAC/VAL on B2 receptor and eNOS expression in the cortex after GCI/R injury in mice. Immunohistochemical staining of cortex revealed strong positive expression of BK positive (A) and eNOS positive (B) neurons in the sham and the SAC/VAL treatment groups (30/50 mg/kg and 60/100 mg/kg) when compared to I/R group. Green arrows showing neurons with positive expression while red arrows indicate no/minimal Bk and eNOS positive neurons. The expressing cells were quantified in five randomly selected regions of cortex (magnification 400x, scale bar 50 μm) and represented as percentage of total cells in each group (C, D). Values are expressed as mean ± SEM, ^#p < 0.05 versus sham, ***p < 0.001, **p < 0.01, *p < 0.05 versus I/R group, ^$p < 0.05 versus SAC/VAL (60/100 mg/kg) group.

DISCUSSION

In the present study, we demonstrated that combined treatment with SAC and VAL significantly improved neurological deficits, reduced oxidative stress and neuroinflammation, and reversed the neuronal damage in the mice subjected to GCI/R injury. We further found that pretreatment with SAC/VAL significantly increased the expression of Bradykinin B2 receptors and eNOS proteins when examined through IHC. Intriguingly, these neuroprotective effects of SAC/VAL were curtailed in the presence of bradykinin B2 receptor antagonist, ICT, which substantiates our hypothesis that increasing the level of endogenous bradykinin and activating its signaling pathway, thereby improving the cerebral blood flow in the ischemic area, may be responsible for the neuroprotective potential of neprilysin inhibitor.

SAC/VAL, the first angiotensin receptor-neprilysin inhibitor (ARNI) has been approved by the FDA for the treatment of heart failure with reduced ejection fraction.³¹ Consistent with previous research, the present study employed the combination of sacubitril and valsartan due to their complementary mechanism, offering broader neuroprotection in cerebral ischemia. Sacubitril enhances natriuretic peptide with vasodilatory and antioxidant effects, while valsartan prevents angiotensin-II-mediated damage.³² A previous study demonstrated that combination therapy, LCZ696, was more effective in reducing ischemic injury compared with valsartan alone, likely due to the additional protective role of elevated natriuretic peptides.²⁰

Peptidase activity plays a major role in the tissue-specific regulation of bradykinin levels.³³ Inhibition of neprilysin, a membrane-bound metallopeptidase, increases the bradykinin level by inhibiting its degradation.³⁴ Several studies indicate that the administration of neprilysin inhibitor has increased the cyclic GMP and bradykinin levels in kidney tubules and the heart, which in turn mediates biological effects such as vasodilation, natriuresis, diuresis, and hypotension.³⁵ Many studies have utilized bradykinin antagonists to elucidate the role of bradykinin in mediating the effects of neprilysin inhibitors. ICT, a bradykinin 2 receptor antagonist (B2R), effectively reduced the ischemia-reperfusion injury caused by neprilysin-inhibitor in the rat heart.³⁶ Moreover, ICT inhibited the production of nitric oxide stimulated by neprilysin inhibitors in isolated canine coronary microvessels.³⁷ The B2R, activated by the endogenous ligand, plays a key role in neurogenesis, neural differentiation, and inflammation regulation. B2R has been demonstrated to protect neurons from ischemia-reperfusion injury through modulating multiple signaling pathways.^38–40 A substantial body of literature acknowledges the complex nature of bradykinin in neuroprotection; however, the therapeutic implications of neprilysin inhibitors in cerebral ischemia remain unclear and require further investigation.

In the present study, the bilateral carotid artery occlusion model was used to induce global cerebral ischemia in mice. The hippocampus and cortex both are highly vulnerable to ischemic insults induced by BCCAO, exhibiting cognitive and neuromuscular impairment in mice.^23,41 In this study, muscle coordination was assessed using a beam walk test, while memory impairment through Y-maze and passive avoidance test targeting spatial working and non-spatial memory, respectively. In line with the previous studies, BCCAO-induced GCI/R injury significantly impaired learning and memory in mice.²⁵ However, SAC/VAL-treated groups exhibited less spatial memory deficits demonstrated by increased entries and spontaneous alternation behavior in the Y-maze paradigm. Additionally, the SAC/VAL-treated group improved memory retrieval, evidenced by increased step-through latency in the passive avoidance test. SAC/VAL at 60/100 mg/kg produced more pronounced cognitive benefits, while concurrent administration of SAC/VAL at both doses significantly enhanced muscle coordination, as indicated by reduced number of foot slips and time taken to traverse the narrow beam. Consistent with these findings, Ashram et al. demonstrated in their recent studies that SAC/VAL treatment has significantly improved spatial memory performance in diabetic rats.³² These data were further supported by histopathological assessment of the hippocampus and cortex, where mice subjected to GCI/R injury showed several degenerated neurons with marked pyknosis indicating severe ischemic injury.⁴² Besides, we observed that SAC/VAL treatment significantly prevented neuronal loss in these regions and showed more integrity in the neuronal structure. Cites in experimental studies showed that bradykinin preserved spatial memory and promoted functional recovery in mice after an ischemic stroke, as indicated by a decrease in infarct size, preservation of the blood–brain barrier and reduction in inflammation and oxidative stress. Simultaneously, blocking B2 receptors with the antagonist HOE140 reversed these protective effects.^43,44 These findings are consistent with the results presented herein.

Oxidative stress and neuroinflammation are closely associated with ischemia-induced neurodegeneration in stroke patients. Neurons that are susceptible to free radical damage during reperfusion have been reported to exhibit lipid peroxidation. Evidence shows increased MDA levels and decreased GSH following bilateral carotid artery occlusion, indicating oxidative stress after an ischemic event, which is consistent with our results.⁴⁵ Also, the occurrence of oxidative stress is strongly linked to the activation of cytokines in acute ischemic stroke.⁴⁶ NFκB plays a central role in neuroinflammation by promoting the production of cytokine storms and contributes to the progression of stroke.⁴⁷ Our results demonstrated that SAC/VAL pretreatment attenuated the cytokine storm induced by GCI/R, as evidenced by reduced brain levels of TNF-α and IL-6. Immunohistochemical analysis revealed suppressed NFκB nuclear translocation, supporting its anti-inflammatory potential. Additionally, SAC/VAL reduced oxidative stress, indicated by lower MDA levels and increased GSH and catalase activity. Although several studies suggest that BK can trigger inflammatory responses in the CNS, it has also been shown to exert anti-inflammatory and neuroprotective effects.¹⁰ Yang et al. reported that BK may indirectly reduce inflammation and improve hindlimb function in a spinal cord ischemia-reperfusion injury rat model by modulating the NFκB signaling pathway, which was consistent with the results from an in vitro model using microglia cells.⁴⁸ Similarly, our data validated that combined treatment with SAC/VAL restrained the proinflammatory response, leading to reduced I/R injury, possibly mediated through the activation of B2R and its associated signaling pathways.

Bradykinin has been shown to mediate vascular relaxation through eNOS activation, promoting NO release and protecting against cellular senescence. Wang et al demonstrated that the PI3K/Akt/eNOS signaling pathway plays a role in neuroprotection in H₂O₂-induced brain injury.⁴⁹ Consistent with these findings, our study demonstrated that the SAC/VAL pretreatment upregulated B2R and eNOS protein expression, as confirmed by immunohistochemical analysis. Notably, coadministration of ICT, a B2R antagonist, reduced eNOS expression, suggesting sacubitril’s neuroprotective effects are likely mediated by the endogenous generation of kinin and B2R-dependent activation of eNOS-induced vasodilation. This study takes a foundational approach to sacubitril/valsartan’s neuroprotective effects and has certain limitations. Comprehensive mechanistic studies are needed to understand how sacubitril and valsartan individually interact with bradykinin and related signaling pathways. Additionally, further investigations focusing on glia-mediated inflammation will help strengthen the evidence for its proposed anti-inflammatory benefits. Finally, employing the clinical 1:1 ratio of SAC/VAL in future studies will be important for assessing the translational relevance of the observed neuroprotective outcomes.

CONCLUSIONS

The present study reveals that treatment with sacubitril–valsartan promotes functional recovery in mice following global cerebral ischemia-reperfusion injury by improving neurological impairment and reducing inflammatory response and oxidative stress. Additionally, our results suggest that the neuroprotective effects of SAC/VALmay be associated with the endogenously produced bradykinin and the subsequent activation of B2R and eNOS. Simultaneously, the B2R antagonist, ICT could counteract the neuroprotective effects of SAC/VAL in mice with ischemic injury. This study collectively provides an essential framework for understanding the relationship between neprilysin inhibition and B2R as a promising target for therapeutic approaches in stroke.

AUTHORS’ CONTRIBUTIONS

G.M.: Writing an original draft, methodology, investigation, data curation, and validation. M.S.: Review and editing, visualization, supervision, conceptualization, and resources. A.K.N.: Review and supervision, conceptualization, resources.

Footnotes

ACKNOWLEDGMENTS

The authors express their sincere gratitude to Raj Kumar Goel Institute of Technology (Pharmacy), Ghaziabad, India, for their invaluable support for the successful execution of this study.

DISCLOSURE STATEMENT

The authors report that there are no competing interests to declare.

FUNDING INFORMATION

No funding was received for this article.

Abbreviations Used

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