Introduction

Stroke is the second leading cause of death worldwide1. It is characterized by high incidence, high recurrence rate, high complication rate and high disability rate2. Epidemiological studies show that the estimated prevalence, incidence, and mortality rate of stroke in China in 2020 were 2.6%, 505.2 per 100 000 person-years, and 343.4 per 100 000 person-years, respectively3. The incidence of stroke is increasing year by year, especially ischemic stroke (also known as cerebral infarction, CI). It is necessary to find strategies to improve stroke prevention in the general population of China.

Atherosclerosis (AS) is a systemic disease of blood vessels, which causes the thickening of the blood vessel lining or the formation of plaques that narrow or block the blood vessel cavity4. Intracranial arterial system is also a common site of AS. The stenosis and occlusion of cerebral arterial system caused by AS is one of the main causes of stroke, while intracranial atherosclerotic stenosis (ICAS) and occlusion is the main cause of CI5. A large number of studies have confirmed that there are racial differences in the distribution of arterial stenosis and occlusion sites, and intracranial artery stenosis is more common in Asian population than extracranial carotid artery stenosis6, which may be related to environmental and genetic factors7. Therefore, the stenosis of intracranial artery system has become an important cause of CI in China.

C-reactive protein (CRP) and albumin are not only markers of systemic inflammation, but also active participants in the formation and destruction of atherosclerosis8. CRP is a common inflammatory factor, and its impact on cardiovascular and cerebrovascular arteriosclerotic diseases is controversial9;10. Studies have shown that a high level of CRP is significantly correlated with intracranial artery stenosis11. However, other studies have suggested that CRP does not increase the risk of intracranial artery stenosis in the population12;13. Human blood albumin can provide nutrients for the body tissues and cells of CI patients, help to reduce brain edema and blood stasis, improve the condition and accelerate the recovery of the disease14. The CRP/ albumin ratio is a valuable combination of inflammatory biomarkers that can be used to accurately predict periprosthetic joint infections15. Given that both markers are influenced by inflammatory processes and nutritional status, the CRP/ albumin ratio serves as an integrated measure, offering insight into the delicate balance between these factors. This ratio has been increasingly recognized as a useful tool for evaluating cardiovascular and cerebrovascular risks, making it highly relevant in our study of intracranial atherosclerotic stenosis. Therefore, we aimed to provide basis for early diagnosis and treatment of CI by exploring the correlation between CRP/ albumin ratio and degree of ICAS.

Materials and methods

Research object

We retrospectively analyzed the degree of stenosis in patients with CI admitted to the neurology department of the Dongguan Tungwah Hospital, Dongguan, China and Dongguan Songshan Lake Tungwah Hospital, Dongguan, China from February 2021 to October 2022. Inclusion criteria: (1) magnetic resonance imaging (MRI) confirmed the diagnosis of CI; (2) complete computed tomography angiography (CTA) examination. Exclusion criteria: (1) patients with local/systemic inflammatory diseases (acute/chronic), active infections, hemorrhagic CI, malignant tumors, autoimmune diseases, blood system diseases; (2) patients with severe cardiopulmonary, liver and kidney dysfunction; (3) patients with incomplete laboratory examination or imaging examination; (4) CI caused by other causes except AS.

General clinical data and related risk factors for cerebrovascular disease were collected. The main indicators included gender, age, hypertension, diabetes, hyperlipidemia, family history of cerebrovascular disease, smoking and drinking history, and glycosylated hemoglobin, homocysteine, blood glucose, total cholesterol, triglyceride, high-density lipoprotein, CRP, albumin and other indicators in hematology examination.

Sample size justification

A power analysis was conducted to determine the appropriate sample size for this study. Based on an expected effect size of 0.5 (d, considered a medium effect size), a power of 0.8 (β, 80% probability of detecting an effect if one exists), and an alpha level of 0.05 (α, significance level), a standard deviation of 1 (σ), the required sample size was calculated using this fomular:

$$n = \frac{{2 \times \left( {Z_{{\alpha /2}} + Z_{\beta } } \right)^{2} \times \sigma ^{2} }}{{{\text{d}}^{{\text{2}}} }}$$

Diagnosis and grouping of ICAS

During hospitalization, the patient underwenthead and neck MRI examination using Sienens Avanto 3.0T superconducting MRI machine. The main imaging protocols for MRI examination include T1-weighted imaging, T2-weighted imaging, diffusion weigh imaging (DWI) and CTA. The size of the CI lesion in the patient was determined by the maximum lesion diameter displayed on DWI. If the vascular lumen is smooth and uniform in thickness, there is no stenosis in this segment of blood vessels. If the blood flow signal is lost, the lumen segmental thinning or uneven thickness, or even missing, the blood vessel is considered to have stenosis. Patients were grouped according to the degree of stenosis: low group (n = 104): no stenosis and mild stenosis (less than 50%); high group (n = 124): moderate stenosis (50–70%) and severe stenosis (greater than 70%).

Microarray expression profiling

Microarray profile GSE202518 and GSE180470 of CI related gene were downloaded from the Gene Expression Omnibus (GEO) database (http://www.ncbi.nlm.nih.gov/geo). All differentially expressed genes (DEGs) of two groups were analyzed by an online analysis program Geo2R provided by GEO database. P < 0.05 and |log2FC| ≥ 1.5 is the standard of screening DEGs.

RT-qPCR

RT-qPCR has been used to determine the mRNA levels of TMTC1 in serum. Total RNA was extracted by TRIzol (Invitrogen, USA) and cDNA was synthesized by High-Capacity cDNA Reverse Transcription Kit (Qiagen, USA). Then the absorbance value of cDNA solution at 260 nm was measured by ultraviolet spectrophotometer (Shimadzu, Japan) and the concentration of cDNA template was converted. PCR was performed using an ABI-7500 FAST Real-time PCR platform at 95 ℃ for 20 s, followed by 95 ℃ 3 s and 60 ℃ 30 s for the next 40 cycles. U6 was used as internal reference, 3 multiple holes were set for each. 2−ΔΔCt was used to indicate the relative expression level of TMTC1. TMTC1 upstream primer: 5’-CTGTCTTCAAGAATCGTGGACTT-3’, downstream primer: 5’-CCGCCTCAGTATGAATAGGATGT-3’.

Statistical analysis

SPSS version 26.0 (IBM, USA) has been used to analyze all data. The Student t-test analysis was on data that conforms to normal distribution, and the data were represented as mean ± standard deviation (SD). Data that does not follow a normal distribution were analyzed using rank sum test, and data were represented using median and interquartile range. In addition, correlation analysis was conducted using chi square test. To assess the relationship between CRP/albumin ratio, TMTC1 expression, and the degree of ICAS, binary logistic regression models were used. The models were adjusted for potential confounders. The combined use of CRP, CRP/albumin ratio and TMTC1 expression was also evaluated in receiver operating characteristics (ROC) analysis, and the corresponding AUC, sensitivity, and specificity with 95% CIs were reported. p < 0.05 was considered as significant.

Results

High ratio of CRP and albumin is linked to the degree of ICAS

To ensure statistical validity and account for potential dropouts, we included 228 participants in the study, which exceeds the required sample size and ensures that the study is adequately powered to detect meaningful effects. A total of 104 patients with low stenosis and 124 patients with high stenosis group were enrolled in this study. Age, medical history and other parameters were not statistically different between the two groups, while blood glucose, CRP and CRP/ albumin ratio were significantly up-regulated in the high stenosis group (*p < 0.05, Table 1). Excluding factors with no significant differences according to Table 1, binary regression analysis was conducted on HCY, Blood glucose, CRP, CRP/ albumin ratio, and TMTC1, and the predicted probabilities were analyzed using ROC curve analysis. The results suggested that the impact of CRP/ albumin ratio and TMTC1 on the results were statistically significant (Table 2). These factors combined have a high sensitivity in distinguishing the degree of stenosis (Figure S1, Table 3, p < 0.001, AUC = 0.767 with 95% CI, 0.706 to 0.829). As indicated in Fig. 1A, the ratio of CRP/ albumin was significantly higher in the high group with patients with sever stenosis (p < 0.001, Fig. 1A). Next, ROC curve analysis was performed to evaluate the sensitivity and specificity of CRP/ albumin ratio for the diagnosis of ICAS. The results indicated that the area under the curve (AUC) of CRP/ albumin ratio was 0.672 (p < 0.001, AUC = 0.672 with 95% CI, 0.602 to 0.741) (Fig. 1B; Table 4), suggesting good diagnostic accuracy. When CRP was detected alone, the expression were significantly increased in the high group (Figure S2A), but the ROC curve results indicated statistical difference (p < 0.001,, AUC = 0.679 with 95% CI, 0.610 to 0.747) (Figure S2B). Compared with the CRP/ albumin ratio indicator, its specificity is not significant.

Table 1 Comparison of intracranial atherosclerotic stenosis degree.
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Table 2 Logistic regression analysis.
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Table 3 ROC curve analysis after adjusting confounding factors.
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Fig. 1
figure 1

High ratio of CRP and albumin is linked to the degree of ICAS. (A) The level of CRP/ albumin in serum of patients with ICAS. (B) The ROC curve of CRP/ albumin.

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Table 4 ROC curve for predicting intracranial atherosclerotic stenosis.
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Correlation between TMTC1 level and prognosis and diagnose of ICAS

Afterwards, two microarray profile GSE202518 and GSE180470 of CI related genes were identified. Top 100 up-regulated genes were screened respectively, and the Venn diagram analysis suggested that TMTC1 was highly expressed in CI (Fig. 2A). The mRNA expression of TMTC1 was significantly elevated in high stenosis group (p < 0.0001, Fig. 2B), and the ROC curve analysis was performed to evaluate the sensitivity and specificity of TMTC1 for the diagnosis of ICAS. The results indicated that TMTC1 has significant sensitivity and specificity in diagnosing ICAS (p < 0.001, AUC = 0.684 with 95% CI, 0.615 to 0.753) (Fig. 2C; Table 4).

Fig. 2
figure 2

Correlation between TMTC1 level and prognosis and diagnose of ICAS. (A) The venn diagram of the top 100 upregulated genes in ischemic stroke from two microarray profile GSE202518 and GSE180470. (B) The mRNA level of TMTC1 in serum of patients with ICAS. (C) The ROC curve of TMTC1.

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Combination of CRP/ albumin ratio and TMTC1 expression may sensitive biomarkers for the diagnosis of ICAS

Subsequently, the ROC curve analysis was performed to evaluate the sensitivity and specificity of combination of CRP/ albumin ratio and TMTC1 expression for the diagnosis of ICAS. The results indicated that combination of CRP/ albumin ratio and TMTC1 has significant sensitivity and specificity in diagnosing ICAS (p < 0.001, AUC = 0.724 with 95% CI, 0.659 to 0.790) (Fig. 3; Table 4).

Fig. 3
figure 3

The ROC curve analysis was performed to evaluate the sensitivity and specificity of combination of CRP/ albumin ratio and TMTC1 expression for the diagnosis of ICAS.

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Discussion

In this study, patients with CI were divided into low artery stenosis group and high artery stenosis group, and the results showed that 51.6% of patients in the high artery stenosis group. The ratio of CRP/ albumin was significantly higher in the high group with patients with sever stenosis. Meanwhile, TMTC1 was identified as a gene highly expressed in high artery stenosis group. These two indicators may be potential markers for the prognosis and diagnosis of ICAS, and the combined application of CRP/ albumin ratio and TMTC1 expression improves the sensitivity and specificity of ICAS diagnosis.

AS is the most common cause of CI and an important pathological basis of CI. Epidemiological studies suggest that the incidence of intracranial atherosclerosis is higher than that of extracranial artery in Asian population, and intracranial atherosclerosis is the most important etiological subtype of CI in China16. Among the many factors causing intracranial artery stenosis, AS is considered to be the most common factor, and ICAS is also an important cause of CI6. Wong et al. performed TCD examination on 705 Chinese patients hospitalized for acute stroke, and found that 36.6% of patients had intracranial vascular lesions, and the distribution of intracranial vascular stenosis in patients was successively middle cerebral artery (73.3%) vertebrobasilar artery (40.3%) and anterior cerebral artery (35.9%)17. AS plaques are more likely to appear at the bifurcation or curvature of arteries and the branch opening of arteries. Therefore, intracranial atherosclerosis stenosis is more likely to occur in the siphon segment, the rock segment, the cavernous sinus segment and the middle cerebral artery of the internal carotid artery. The clinical symptoms, prognosis and treatment of CI patients due to anterior and posterior circulatory artery stenosis are not identical. Therefore, the analysis of the distribution characteristics of intracranial artery stenosis in CI patients is of great significance for the selection of clinical treatment and the evaluation of prognosis.

Previous studies have shown that compared to subjects without ICAS, ICAS patients have significantly higher levels of white blood cells, neutrophils, neutrophil/lymphocyte ratio, low-density lipoprotein, non high-density lipoprotein, CRP, high-sensitivity CRP, uric acid, creatinine, and homocysteine, but lower levels of lymphocytes8. Another study found that hsa_circ_0003574 is significantly upregulated in ICAS patients, which may be related to ischemic stroke caused by ICAS18. The detection of these biomarkers may help stratify ICAS risk in stroke free subjects for neurovascular examination and further prevention strategies in high-risk subjects to prevent stroke or vascular events. CRP is an acute phase protein whose levels increase under conditions of infection, ischemia, trauma, and other inflammatory conditions19. Albumin is the main protein in plasma, and its level is negatively correlated with nutritional status and inflammation. The CRP/ albumin ratio has been proposed as a predictive biomarker that may reflect the complex interaction between inflammation and nutritional status19. Recent meta-analyses and studies have highlighted the importance of systemic inflammation in AS, with CRP and albumin being commonly studied as key biomarkers for vascular health20,21,22. Previous research has demonstrated that high CRP levels are associated with poor outcomes in cardiovascular and cerebrovascular diseases23;24, while albumin has been linked to inflammation and nutritional status, both of which are crucial in the progression of AS25;26. In the context of ICAS, this ratio may be related to the progression of vascular inflammation and atherosclerosis, which may explain its association with the degree of stenosis.

The CRP/ albumin ratio is a valuable combination of inflammatory biomarkers that can be used to accurately predict periprosthetic joint infections15. In this study, high ratio of CRP and albumin is linked to the degree of ICAS. However, the sensitivity of CRP/ albumin ratio was not high enough. Afterwards, the highly expressed TMTC1 gene was predicted to be a potential biomarker for ICAS. TMTC1 encodes the transmembrane O-mannose-transferase targeting cadherin, which is mainly located in the endoplasmic reticulum and is involved in regulating the calcium homeostasis of the endoplasmic reticulum27. TMTC1 has been reported to promotes invasiveness of ovarian cancer cells28. Highly expressed TMTC1may be a promising biomarker for osteogenic differentiation mesenchymal stem cells29. Interestingly, the TMTC1 gene is widely expressed in the brain and has been shown to be a risk gene for schizophrenia30;31. The combination of CRP/ albumin ratio and TMTC1 levels increased the sensitivity for ICAS diagnosis. In conclusion, CRP/ albumin ratio has important clinical significance in the diagnosis and prognosis evaluation of acute CI, especially in patients with ICAS. Meanwhile, the expression of TMTC1 may also provide additional biomarker support for the diagnosis of ICAS.

The cost-effectiveness of combining the CRP/ albumin ratio and TMTC1 expression as biomarkers for diagnosing ICAS is worth exploring. The CRP/ albumin ratio, being a routine inflammatory marker, is simple to measure and low-cost, making it easy to incorporate into daily clinical screenings for initial assessment. However, the detection of TMTC1 requires more complex laboratory techniques (such as RT-qPCR), which increases the cost and time of testing, potentially limiting its widespread use in resource-limited settings. Nevertheless, in large hospitals or specialized centers, combining these two markers could improve diagnostic accuracy, enable early detection of ICAS, and reduce long-term healthcare costs. Therefore, it is suggested that the CRP/ albumin ratio be used for initial screening, with TMTC1 serving as an adjunct for further confirmation. Future economic studies should evaluate the cost-effectiveness of this combined biomarker approach in different healthcare settings to provide clearer guidance for clinical implementation.

There are some limitations to this study. First, this study is retrospective and cannot determine the causal relationship between CRP/ albumin and TMTC1. Second, it is an observational study and there may be some unmeasured or residual confounding effects. Third, patients are from a single stroke center and there may be selection bias. Therefore, the results still need to be further verified by multi-center studies with larger sample sizes. Future research should focus on validating these findings in larger multicenter cohorts. This will help evaluate the clinical efficacy of biomarker combinations in different populations and medical environments, and further confirm their value in practical clinical practice.

Conclusion

High levels of CRP/ albumin ratio and TMTC1 expression were associated with the degree of ICAS, and may be potential diagnostic and prognostic markers for the ICAS.