Impact of Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9) Inhibitor on Immune-Inflammatory Responses in the Acute Phase of Ischemic Stroke

Introduction

Stroke remains one of the leading global causes of mortality and long-term disability. Based on etiology, stroke is broadly classified into ischemic stroke and hemorrhagic stroke, with ischemic stroke accounting for 80–85% of all stroke cases.¹ Acute ischemic stroke (AIS) involves complex pathophysiological mechanisms and induces systemic immune alteration.²

During the acute phase of AIS, substantial amounts of inflammatory cytokines such as interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) are released from damaged brain tissue and vasculature.^3–5 These mediators promote infiltration of peripheral neutrophils and monocytes/macrophages into ischemic regions. Importantly, AIS causes lymphopenia, mainly reducing circulating CD4-positive (CD4⁺) T lymphocytes, which significantly increases susceptibility to infections.^6,7 Additionally, peripheral immune cells including T lymphocytes and B lymphocytes may exacerbate neuronal damage by triggering central nervous system inflammatory cascades and disrupting cerebral microenvironment homeostasis.⁸ Therefore, targeting inflammation is one of the key directions for AIS intervention. Our team’s previous research has confirmed that edaravone dexborneol, a neuroprotective agent, exerts a distinct anti-inflammatory effect in AIS. It inhibits the expression of proinflammatory factors and upregulates the levels of anti-inflammatory factors. Meanwhile, this agent can improve patients’ functional outcomes 90 days after stroke onset.⁹

Extensive research has established dyslipidemia, particularly elevated low-density lipoprotein cholesterol (LDL-C) levels, as a major risk factor for intracranial atherosclerotic stroke occurrence and recurrence.¹⁰ Although current clinical guidelines recommend statins as foundational lipid-lowering therapy with target LDL-C levels < 1.8 mmol/L or ≥ 50% reduction from baseline, achievement of these lipid goals remains suboptimal in clinical practice.¹¹ It warrants emphasis that despite demonstrating anti-inflammatory properties (such as reducing high-sensitive C-reactive protein (hs-CRP) levels),¹² high-intensity statin therapy fails to fully eliminate residual inflammatory risk in AIS patients.^13,14

Recent developments highlight proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors as a novel therapeutic approach. These agents not only substantially reduce plasma LDL-C via upregulation of LDL receptor (LDL-R) pathways but may also directly modulate immune-inflammatory responses.¹⁵ Preclinical studies demonstrate that PCSK9 deficiency suppresses NOD-like receptor protein 3 (NLRP3) inflammasome activation and reduces proinflammatory cytokine release (including interleukin-18 (IL-18) and TNF-α) following cerebral ischemia.¹⁶ Moreover, PCSK9 inhibitors exhibit anti-inflammatory effects by inhibiting Toll-like receptor 4/nuclear factor-kappa B (TLR4/NF-κB) signaling transduction in various disease models.^16–18 Clinical evidence suggests that PCSK9 monoclonal antibodies provide enhanced cardiovascular benefits particularly for patients with elevated baseline hs-CRP levels.¹⁹ Furthermore, PCSK9 inhibitors exert anti-inflammatory effects potentially independent of their lipid-lowering properties. Marfella et al demonstrated through human carotid plaque analysis that PCSK9 inhibitors significantly reduced pro-inflammatory protein expression (including NLRP3, IL-1β) while increasing anti-inflammatory Sirtuin 3 (SIRT3) and collagen content within plaques. These changes happened even in patients with LDL-C levels below 100 mg/dL.²⁰

Notably, statins exert immunomodulatory effects on peripheral immunity through multiple mechanisms. Statins primarily inhibit the mevalonate pathway, reducing isoprenoid intermediate production, thereby downregulating T lymphocyte activation markers including major histocompatibility complex class II molecules and CD40 ligand.^21,22 Additionally, statins regulate T helper 1/T helper 2 (Th1/Th2) lymphocyte balance by suppressing proinflammatory Th1 responses while enhancing anti-inflammatory Th2 polarization.^23–25 Recent studies show that PCSK9 regulates the growth, development, and death of multiple T cell types. These include CD4⁺ T cells and CD8-positive T cells (CD8⁺ T cells), cytotoxic T lymphocytes (CTLs), and regulatory T cells (Tregs). Blocking PCSK9 in tumor cells may boost anti-tumor effects by activating CD8⁺ T cells.²⁶ One key mechanism involves PCSK9 binding to major histocompatibility complex class I (MHC-I). This binding triggers lysosomal degradation of MHC-I, reducing its surface display on tumor cells.²⁷ Liu’s team demonstrated PCSK9’s immune effects in atherosclerosis plaques. Silencing PCSK9 reduced harmful inflammation by Th1 and Th17 cells. These findings suggest that PCSK9 inhibitors might help fight heart disease not just by lowering LDL cholesterol, but also through direct immune effects.²⁸ However, substantial knowledge gaps persist regarding PCSK9 inhibitors’ direct effects on peripheral blood lymphocytes. Furthermore, research investigating PCSK9 inhibitors’ impact on acute-phase immune-inflammatory responses following human AIS remains limited. Large-scale clinical trials such as FOURIER and ODYSSEY OUTCOMES primarily focus on long-term cardiovascular outcomes, leaving unanswered questions about these agents’ capacity to mitigate early post-stroke immune dysregulation occurring within days of stroke onset.

To address these critical knowledge gaps, we designed a prospective study to evaluate the effects of combination therapy with PCSK9 inhibitors and statins compared to statin monotherapy on early peripheral blood inflammation and immunomodulation in AIS patients with large artery atherosclerosis (LAA).

Methods

This study consecutively enrolled patients with AIS from the prospective stroke registry of Nanjing First Hospital Affiliated to Nanjing Medical University between November 2024 and May 2025. Participants were derived from AISDTS (www.clinicaltrials.gov, NCT05410457) and sICASBLM (www.clinicaltrials.gov, NCT05397405). The study protocol was approved by the Ethics Committee of Nanjing First Hospital (No. KY20220518-03-KS-01 and No. KY20220518-04-KS-01) and strictly adhered to the ethical principles of the Declaration of Helsinki. Written informed consent was obtained from all enrolled patients or their legal guardians. This observational study used routinely collected health data. To ensure transparent and complete reporting, we followed the RECORD (REporting of studies Conducted using Observational Routinely collected health Data) statement.

In this study, the inclusion criteria were as shown as follows: (1) aged 18 to 85 years; (2) patients hospitalized with non-cardioembolic AIS diagnosed within 14 days of symptom onset; (3) AIS confirmed by computed tomography (CT) or magnetic resonance imaging (MRI) and vascular imaging (eg., computed tomography angiography (CTA) or magnetic resonance angiography (MRA)) demonstrating stenosis ≥50% or unstable plaque in the intracranial culprit vessel, consistent with a diagnosis of LAA; (4) LDL-C levels ≥ 2.6 mmol/L or ≥ 1.4 mmol/L despite long-term statin therapy. The exclusion criteria were as follows: (1) definite cardioembolic sources (eg., chronic/paroxysmal atrial fibrillation, endocarditis, intracardiac thrombi, or vegetations); (2) intracranial hemorrhage, mass lesions, or traumatic brain injury detected on admission CT; (3) active infection diagnosed within 1 week before AIS onset or during hospitalization (eg., pneumonia, urinary tract infection, fever of unknown origin); (4) neurological dysfunction secondary to malignancy; (5) severe multiorgan dysfunction; (6) hematological disorders or autoimmune diseases; (7) premorbid modified Rankin Scale (mRS) score >2; (8) PCSK9 inhibitors use within 6 months prior to enrollment; (9) pregnancy, lactation, or planned pregnancy; (10) patients who received intravenous thrombolysis, endovascular intervention (including mechanical thrombectomy, angioplasty, etc.); (11) incomplete clinical data or loss to follow-up.

According to clinical recommendations and patient preferences, participants were assigned to two groups without randomization: the standard treatment group received atorvastatin at a dosage of 40 mg daily; the intensive treatment group received atorvastatin at 40 mg daily plus subcutaneous injections of the PCSK9 inhibitor evolocumab 140 mg every two weeks. All patients received standardized antiplatelet therapy according to previously published protocols, specifically dual antiplatelet therapy with aspirin 100 mg/d combined with clopidogrel 75 mg/d.²⁹ For patients at high bleeding risk or those who experienced bleeding after medication, single antiplatelet therapy was adopted after thorough communication of the relevant risks and benefits with the patients and their families.

All enrolled patients underwent comprehensive clinical evaluations, including demographic data, stroke risk factors, white matter hyperintensity (WMH) burden, clinical symptom scoring (admission National Institutes of Health Stroke Scale (NIHSS) and premorbid mRS), and laboratory testing (fasting blood samples collected on day 2 and 2-week follow-up). Since random venous blood glucose was not available immediately after admission in most patients, we assessed glycemic status using morning fasting blood glucose on the second day of admission. Hyperglycemia was defined as fasting blood glucose ≥7.0 mmol/L on the second day after admission, a threshold widely recognized as one of the diagnostic criteria for stress hyperglycemia in the setting of acute ischemic stroke.³⁰ WMH burden was assessed using the modified Fazekas 3‑point scale.³¹ We combined grades 1 and 2 (mild to moderate) into one group and compared them with grade 3 (severe). Lipid profiling included total cholesterol (TC), triglycerides (TG), LDL-C, high-density lipoprotein cholesterol (HDL-C), and remnant cholesterol (RC), along with measurements of hs-CRP, lipoprotein-associated phospholipase A2 (Lp-PLA2) levels, white blood cell (WBC) count, lymphocyte count, monocyte count, neutrophil count, and blood platelet count. Calculated immune-inflammatory indices included neutrophil-to-lymphocyte ratio (NLR), monocyte-to-lymphocyte ratio (MLR), platelet-to-lymphocyte ratio (PLR), white blood cell-to-lymphocyte ratio (WLR), and systemic immune-inflammation index (SII). Additionally, at baseline and the 2-week follow-up, 5 mL of peripheral venous blood anticoagulated with EDTA was collected for quantitative analysis of 12 cytokines and lymphocyte subsets. The 12 cytokines, including IL-1β, Interleukin-2 (IL-2), Interleukin-4 (IL-4), Interleukin-5 (IL-5), IL-6, Interleukin-8 (IL-8), Interleukin-10 (IL-10), Interleukin-12p70 (IL-12p70), Interleukin-17 (IL-17), TNF-α, Interferon-gamma (IFN-γ), Interferon-alpha (IFN-α), were measured using multiplex bead-based flow immunofluorescence with a commercial kit (Qingdao Ruiscell Biotechnology Co., Ltd.) according to the manufacturer’s standard protocol. Lymphocyte subsets were analyzed by flow cytometry using reagents from Tianjin Quanbo Tongsheng Biotechnology Co., Ltd., following the manufacturer’s instructions. All samples were finally detected and recorded on a flow cytometer (BD FACSCanto II, BD Biosciences, USA). We analyzed lymphocyte subsets including total T cell, helper T cell (CD4⁺ T cell/Th cell), suppressor/cytotoxic T cell (CD8⁺ T cell/Ts cell), CD4⁺/CD8⁺ ratio, natural killer cell (NK cell), B cell, total lymphocyte, with results expressed as percentages.

This was a prospective observational study. Sample size calculation was performed using PASS 15.0 software with a conservative effect size (Cohen’s d = 0.5),^32,33 a two-sided type I error rate of α = 0.05, and a statistical power of 80%. According to clinical practice and patient preference, the allocation ratio was set at 2:1 (intensive treatment group: standard treatment group). The calculation indicated that approximately 55 patients were required in the intensive treatment group and 28 patients in the standard treatment group, with a total calculated sample size of 83 patients. Considering a 10% dropout rate, the planned total sample size was 100 patients (about 66 in the intensive treatment group and 34 in the standard treatment group). Finally, 72 patients were actually included in the final analysis (24 in the standard treatment group and 48 in the intensive treatment group). The statistical power for the primary inflammatory and lipid parameters was generally sufficient to meet the requirements of this study.

All statistical analyses were performed using SPSS version 27.0 (SPSS Inc., Chicago, IL, USA), while GraphPad Prism 9 was used for graphical representation. Categorical variables were expressed as frequencies. The Kolmogorov–Smirnov test assessed data normality; normally distributed continuous variables were presented as mean ± standard deviation (SD), whereas non-normally distributed continuous variables were reported as medians (interquartile range, IQR). Comparisons were made using the chi-square test for categorical variables, Student’s t-test for normally distributed continuous variables, and the Mann–Whitney U-test for non-normally distributed continuous variables. A two-tailed P-value < 0.05 was considered statistically significant in all analyses.

Results

Baseline Demographics of the Patients

In total, 85 patients meeting the inclusion and exclusion criteria were recruited from Nanjing First Hospital for this study between November 2024 and May 2025. Based on clinical recommendations and patient preferences, the participants were divided into two groups: the standard treatment group with 31 cases, and the intensive treatment group with 54 cases. During the follow-up period, some patients were excluded from the study due to reasons such as loss to follow-up, infection occurred after enrollment, and incomplete laboratory examinations. Ultimately, a total of 72 patients (the standard treatment group: 24 cases; the intensive treatment group: 48 cases) were included in the analysis (Figure 1).

Figure 1 Study flow diagram. The flowchart illustrates the enrollment, treatment allocation, and follow-up of patients in the prospective cohort study. Patients were divided into a standard treatment group (high-intensity statin) and an intensive treatment group (PCSK9 inhibitor and high-intensity statin). Exclusions due to loss to follow-up and post-enrollment infections are indicated. The final analyzed cohorts for each group are shown at the bottom. Abbreviation: Hs-CRP, high-sensitive C-reactive protein.

The baseline characteristics of these patients are presented in Table 1. The average age of the participants was 64.68 ± 10.62 years, and 44 (61.11%) of them were male. The patient cohort primarily comprised individuals with mild stroke, indicated by a median NIHSS score of 2 (0–4) at onset, and a median mRS score of 0 (0–0) before enrollment. Patients in the standard treatment group had a higher proportion of coronary artery disease compared to the intensive treatment group (P < 0.050). There were no statistically significant differences between the two groups at baseline concerning age, gender, stroke risk factors, WMH burden, pre-enrollment mRS and onset NIHSS scores (P > 0.050).

Table 1 Baseline Demographics of the Patients

Lipid Levels Between Baseline and the 2-Week Follow-Up

No significant differences in TC, TG, LDL-C, HDL-C and RC levels were observed between the two groups at baseline (P > 0.050) (Table 2). Both groups exhibited reduced median LDL-C levels compared to their pre-treatment values (P < 0.050), indicating the effectiveness of the drug intervention. Significant disparities in TC levels and change from baseline emerged between the groups at the follow-up period (P < 0.001). Furthermore, the reduction of median LDL-C levels differed significantly for both groups (P < 0.001), decreasing from a baseline value of 2.380 (1.850–3.320) mmol/L to 1.845 (1.490–2.200) mmol/L in the standard treatment group, and from 2.530 (1.720–3.190) mmol/L to 0.800 (0.600–1.030) mmol/L in the intensive treatment group (Figure 2). The change from baseline of LDL-C levels was also significantly different between the two groups (P < 0.001), with the intensive treatment group demonstrating a notably greater reduction compared to the standard treatment group. During the 2-week follow-up period, RC levels significantly decreased in both groups, with larger reductions observed in the intensive treatment group (−0.230(−0.368–0.088) mmol/L vs. −0.300(−0.630–0.020) mmol/L), though the between-group difference in change from baseline did not reach statistical significance (P = 0.316).

Table 2 Comparison of Lipid Levels Between Baseline and the 2-Week Follow-Up in Standard and Intensive Treatment Groups

Figure 2 Changes in serum lipid levels in the intensive treatment group and standard treatment group from baseline to 2-week follow-up. The figure consists of two panels showing dynamic changes in serum levels of (A) TC and (B) LDL-C from baseline (Day 0) to 2-week (Day 14) follow-up in both the intensive treatment group and the standard treatment group. Both groups showed significant reductions in TC and LDL-C levels after treatment (P < 0.001). Abbreviations: TC, total cholesterol; LDL-C, low-density lipoprotein cholesterol.

Inflammatory Markers Between Baseline and the 2-Week Follow-Up

Comparative analysis of inflammatory markers revealed no significant differences between the two groups in baseline levels of hs-CRP, leukocyte subsets (WBC, lymphocyte, monocyte, neutrophil, platelet counts), or ratios (NLR, MLR, PLR, WLR, SII) (P > 0.050), except IL-1β, IL-2, IL-4, IL-5, IL-10, IL-12p70, IFN-α (P < 0.050). Notably, IL-1β levels significantly decreased in both groups at 2-week follow-up (baseline: P = 0.050 vs. 2-week: P = 0.026), though the magnitude of change did not differ (P = 0.765). IL-6 exhibited a divergent trend: the standard treatment group showed increased levels (1.595(−0.683–3.300)) pg/mL, while the intensive group declined (−0.300(−2.075–1.290)) pg/mL (P = 0.024). TNF-α reduction was more pronounced in the intensive group (0.000(−1.090–0.000) pg/mL vs. 0.020(−0.190–0.790) pg/mL (P = 0.041). Other cytokines (eg., IL-2, IL-4, IL-5, IL-10, IL-12p70 and IFN-α) demonstrated persistently lower absolute values in the intensive treatment group at baseline and 2-week (P < 0.001) but comparable longitudinal changes (P > 0.050) (Supplementary Figure 1). Lp-PLA2, hs-CRP, leukocyte subsets and ratios remained unaffected by treatment intensity (P > 0.050) (Table 3). Dynamic changes in IL-6 and TNF-α levels from baseline to the 2-week follow-up are presented in Figure 3.

Table 3 Comparison of Inflammatory Markers Between Baseline and the 2-Week Follow-Up in Standard and Intensive Treatment Groups

Figure 3 Changes in key inflammatory cytokines from baseline to 2-week follow-up. The figure consists of two panels showing dynamic changes in serum levels of (A) IL-6 and (B) TNF-α are shown for both the intensive treatment group and the standard treatment group. Change from baseline = 2-week level − baseline level. Statistical significance markers: *P < 0.05, **P < 0.025, indicating that the difference in the magnitude of change between groups is statistically significant (Mann–Whitney U-test). Abbreviations: IL-6, interleukin-6; TNF-α, tumor necrosis factor-α.

Lymphocyte Subsets Between Baseline and the 2-Week Follow-Up

The study results demonstrated no significant differences in baseline lymphocyte subset distributions between the standard treatment group (n = 24) and intensive treatment group (n = 26). Baseline characteristics were consistent with the overall cohort (Supplementary Table 1), with no significant differences between groups (P > 0.050). After 2-week of follow-up, both regimens induced comparable immunological changes: the total T-cell had equivalent decreases (the standard treatment group: −0.615(−3.885–3.103) % vs. the intensive treatment group:-1.535(−5.200–2.600) %, P = 0.846), Th cell displayed parallel reduction (−4.060(−8.223–0.888) % vs. −3.510(−7.700–1.630) %, P = 0.940), Ts cell showed similar changes (0.645(−1.005–3.388) % vs. 0.935(−1.735–2.065) %, P = 0.382), CD4⁺/CD8⁺ ratio decreased similarly (−0.345(−0.713–0.155) vs. −0.170(−0.395–0.080), P = 0.409), NK cell proportions moderately increased (4.070(−0.988–8.678) % vs. 5.345(−1.060–8.340) %, P = 0.938), and B-cell percentages showed significant reduction (−2.690(−4.358–0.553) % vs. −2.640(−4.565–0.493) %, P = 0.884). Total lymphocyte counts exhibited non-significant decline in the standard group (−3.670(−7.688–0.378) %) versus minimal fluctuation in the intensive group (−0.675(−4.598–4.833) %) (P = 0.135). Crucially, all intergroup comparisons of immunological parameter changes demonstrated statistical equivalence (P > 0.050) (Table 4).

Table 4 Comparison of Lymphocyte Subsets Between Baseline and the 2-Week Follow-Up in Standard and Intensive Treatment Groups

Discussions

Our study demonstrated that intensive lipid-lowering therapy with PCSK9 inhibitors plus statins led to significantly greater reductions in LDL-C levels compared to statin monotherapy, with LDL-C decreasing from 2.530(1.720–3.190) mmol/L to 0.800(0.600–1.030) mmol/L in the intensive treatment group versus 2.380(1.850–3.320) mmol/L to 1.845(1.490–2.200) mmol/L in the standard treatment group (P < 0.001). Importantly, this intensive treatment group was associated with favorable changes in inflammatory biomarkers, including a significant decline in IL-6 (−0.300(−2.075–1.290) pg/mL vs. 1.595(−0.683–3.300) pg/mL, P = 0.024) and TNF-α (0.000(−1.090–0.000) pg/mL vs. 0.020(−0.190–0.790) pg/mL, P = 0.041), suggesting potential anti-inflammatory effects beyond lipid modulation. Although lymphocyte subset distributions (such as total T cell, Th cell, Ts cell, CD4⁺/CD8⁺, NK cell, and B cell) showed comparable reductions between groups (P > 0.050), it is notable that the standard treatment group exhibited a pronounced decline in total lymphocyte count (−3.670(−7.688–0.378) %), and the reduction was more attenuated in the intensive treatment group (−0.675(−4.598–4.833) %) (P = 0.135), although the between-group difference did not reach statistical significance.

PCSK9 inhibitors exert their lipid-lowering effects by specifically binding PCSK9 protein, thereby blocking its interaction with LDLR and markedly reducing circulating LDL-C levels.^34,35 Robust evidence from multinational trials (FOURIER and ODYSSEY OUTCOMES) demonstrates that PCSK9 inhibitors achieve additional 50–60% LDL-C reduction in addition to statin therapy.^36–38 Consistent with prior reports, the intensive treatment group attained rapid LDL-C reduction from 2.530 (1.720–3.190) mmol/L to 0.800 (0.600–1.030) mmol/L at 2-week follow-up, whereas the standard treatment group showed only modest decline from 2.380 (1.850–3.320) mmol/L to 1.845 (1.490–2.200) mmol/L (P < 0.001). Moreover, TC reduction proved significantly greater in the intensive treatment group (−2.090 (−2.910 to −0.930) mmol/L, P < 0.001). The rapid and potent LDL-C-lowering advantage over statin monotherapy underscores their pivotal role in intensive lipid management for acute ischemic stroke.

Beyond lipid-lowering, PCSK9 inhibitors exhibit anti-inflammatory and plaque-stabilizing effects.¹⁵ Giunzioni et al demonstrated in mouse models that PCSK9 secreted by macrophages reaches the plasma compartment and the atheroma, and its accumulation in the lesion directly affects plaque composition, independently of serum lipid levels. It increases inflammatory monocyte infiltration by 32%, upregulates TNF-α and IL-1β, and suppresses anti-inflammatory factors IL-10 and Arginase-1.³⁹ This suggests an additional cardiovascular benefit of anti-PCSK9 therapies. Emerging evidence indicates that PCSK9 inhibitors attenuate neuroinflammation by suppressing NF-κB-mediated release of proinflammatory cytokines (IL-6, IL-1β, TNF-α) and activating GPNMB/CD44 signaling, thereby reducing cerebral infarct volume and improving post-ischemic recovery.^40–42 Clinically, early PCSK9 inhibitor therapy reduces residual myocardial inflammation post-myocardial infarction (assessed by 18F-FDG PET/CT) and may mitigate cardiac remodeling.⁴³ During hospitalization for ACS, PCSK9 inhibitors not only potently reduce lipid levels but also rapidly downregulate pro-inflammatory cytokines (eg., IL-1β, IL-6) and partially suppress anti-inflammatory factors (eg., IL-13, IL-4) in peripheral blood.⁴⁴ Notably, Liu et al found that evolocumab combined with statins significantly reduced IL-6 levels and lowered the risk of early neurological deterioration (END) in patients with large artery atherosclerotic stroke (LAA), whereas no significant benefit was observed in those with small vessel occlusion (SVO). These results suggest that PCSK9 inhibitors may attenuate atherosclerosis-related inflammation by suppressing IL-6, thereby reducing END incidence.⁴⁵ Our study similarly observed significant differences in peripheral blood pro-inflammatory markers between the two groups. The intensive treatment group showed a pronounced reduction in IL-6 levels (P = 0.024) and exhibited an even greater decrease in TNF-α (P = 0.041). These findings provide theoretical support for expanding the indication of PCSK9 inhibitors in early-phase AIS intervention, particularly in high-risk populations with concomitant LAA. Further validation through large-sample randomized controlled trials is warranted.

In the dynamic analysis of lymphocyte subsets, we included 50 subjects with complete baseline and 2-week follow-up data. The neuroinflammatory response following AIS exhibits dynamic temporal evolution: it is activated within minutes of ischemia onset and persists for days to weeks. This process primarily involves the infiltration of peripheral immune cells, activation of resident immune cells in the central nervous system, and the release of various cytokines,⁴⁶ notably affecting lymphocyte subsets.^7,8 Basic research demonstrates that aged stroke mice subjected to antibody-mediated depletion of CD4⁺ T cells exhibited significantly reduced levels of pro-inflammatory cytokines IFN-γ and IP-10 in both peripheral blood and brain tissue, along with improved neurological function, although infarct volume remained unchanged. This finding suggests that delayed CD4⁺ T cell-targeted intervention may serve as a potential therapeutic strategy for patients who miss the reperfusion time window.⁴⁷ Clinical studies further reveal distinct peripheral immune signatures in AIS patients, demonstrating that the frequencies of T cells, Th cells, and Ts cells in AIS are declined dramatically at least 14 days after stroke.⁴⁸ Additionally, another clinical investigation found that AIS patients with favorable outcomes (90-day mRS score ≤ 2) exhibited increased circulating T cell percentages along with elevated CD3⁺ and CD4⁺ T cell counts.⁴⁹ Statins reduce T and B cell counts in murine tissues and circulation by inhibiting lymphocyte homing pathways.^50,51 These preclinical findings have been clinically validated, with statin-treated patients exhibiting markedly suppressed T lymphocyte proliferation and decreased peripheral blood lymphocyte levels.^52,53 This evidence suggests that the immunomodulatory effects of statins require careful evaluation in patients with coexisting immune disorders. At the molecular level, multiple basic studies have confirmed that PCSK9 directly regulates T cell proliferation, differentiation, and apoptosis.^26–28 However, systematic evidence remains scarce regarding the impact of PCSK9 inhibitors on early immune responses in AIS patients. Based on this gap, we conducted a prospective study to evaluate the early effects of evolocumab on peripheral blood lymphocyte subsets in AIS patients. The results demonstrated similar trends in lymphocyte subset changes between the intensive and standard treatment groups at the 2-week follow-up. Several factors may explain this observation: First, PCSK9 inhibitors primarily exert protective effects through local anti-inflammatory mechanisms—by reducing oxidized LDL deposition in foam cells and improving vascular wall inflammatory microenvironments—thereby lowering cardiovascular risk,^20,28 rather than inducing systemic immunosuppression. Second, the acute systemic inflammatory response post-stroke may obscure PCSK9 inhibitors’ specific immunomodulatory effects. Third, complex immunoregulatory interactions may exist between PCSK9 inhibitors and statins. Finally, the single timepoint assessment (Day 14) in this study may have limited our ability to comprehensively capture dynamic lymphocyte alterations. Future investigations should employ longitudinal monitoring, collecting samples at critical timepoints to fully delineate lymphocyte immunophenotypic evolution. Although this study did not identify significant effects of PCSK9 inhibitors on peripheral blood T lymphocyte subsets, this negative finding may reflect the specificity and limitations of their immunomodulatory actions. Future studies should integrate multi-omics technologies (such as combined single-cell transcriptomics and flow cytometry analysis) and longer-term dynamic monitoring to further elucidate the precise regulatory nodes of PCSK9 inhibitors within the post-stroke immune-inflammatory network.

The innovation of this study lies in being the first to systematically and more comprehensively evaluate the impact of PCSK9 inhibitors on peripheral inflammation and immune responses during the acute phase in AIS patients due to LAA, thereby addressing the gap in early immunomodulation data from existing large-scale clinical trials (such as FOURIER and ODYSSEY OUTCOMES). Previous research has confirmed that cerebral hypoperfusion and severe white matter hyperintensity is strong predictors of recurrence in watershed infarction,⁵⁴ highlighting the critical role of vascular pathophysiological abnormalities in the prognosis of AIS. This study found that PCSK9 inhibitors not only significantly reduced LDL-C levels but also markedly suppressed the pro-inflammatory cytokines IL-6 and TNF-α, which are closely associated with atherosclerotic plaque instability and vascular inflammation. This therapeutic effect may be important for reducing the recurrence risk of atherosclerotic AIS. Although this study did not directly assess recurrence outcomes, the reductions in LDL-C and inflammatory factors suggest potential long-term benefits.

This study is an observational investigation with a relatively small sample size, which entails several limitations. First, the small sample size may have compromised statistical power. Therefore, further large‑sample randomized controlled trials are warranted to validate the present findings. Second, the single follow-up timepoint restricted the observation of dynamic changes in inflammatory and immune parameters. Future studies should adopt longitudinal monitoring strategies, with specimen collection and analysis at multiple critical timepoints such as days 3, 7, 14, and 28 post-treatment. Thirdly, our findings may be limited to AIS patients with LAA. The effects of PCSK9 inhibitors in other stroke subtypes (eg., small vessel occlusion) warrant further investigation. Additionally, the absence of functional outcome analyses (eg., END, 90-day mRS scores, stroke recurrence) leaves the long-term effects of PCSK9 inhibitors unclear. However, active follow-up is currently underway in this patient cohort to monitor long-term outcomes. Finally, the lack of cerebrospinal fluid or tissue specimens hindered the investigation of central-peripheral immune interactions. Future research should expand the sample size, extend the observation period, and integrate multi-omics technologies to elucidate the precise molecular mechanisms by which PCSK9 inhibitors modulate post-stroke inflammation and immune responses.

Conclusion

In conclusion, early PCSK9 inhibition add-on therapy in AIS patients with LAA significantly enhances lipid reduction and may exhibit specific anti-inflammatory effects by modulating key pro-inflammatory cytokines (eg., IL-6, TNF-α) compared with statin-alone therapy. While its impact on peripheral lymphocyte subsets appears limited, these findings support its potential dual benefit in acute stroke management. Further large-scale studies are warranted to validate its immunomodulatory role and long-term clinical outcomes.