Correlation analysis of carotid plaque in young patients with newly diagnosed type 2 diabetes and platelet-to-lymphocyte ratio and neutrophil–lymphocyte ratio

Objective Platelet-to-lymphocyte ratio (PLR) and neutrophil–lymphocyte ratio (NLR) have been reported to be related to atherosclerosis. The relationship between PLR, NLR, and carotid atherosclerotic plaque in young patients with newly diagnosed type 2 diabetes has not been clinically reported. This study aimed to analyze the relationship between PLR, NLR, and carotid plaques in young patients with newly diagnosed type 2 diabetes. Method The total 268 patients were divided into the intima-media–thickening (IMT) group (116 cases) and plaque group (152 cases). The plaque group was divided into grade I (62 cases), grade II (47 cases), and grade III groups (43 cases). The counts of platelets, neutrophils, and lymphocytes were determined. Results The diagnostic effect of PLR and NLR was evaluated by the receiver-operating characteristic curve (ROC) and areas under the curve (AUC). The PLR and NLR values in the observation group were higher than those in the control group; these two values in the plaque group were also higher than those in the IMT group. The two values in the grade III plaque group were higher than those in grade II. The values in the grade II plaque group were also higher than those in the grade I plaque group. The ROC of PLR and NLR were 0.722 and 0.653, and the AUC of PLR and NLR were 111.086 and 2.240. PLR yielded a sensitivity of 0.789 and a specificity of 0.612. Neutrophil–lymphocyte ratio yielded a sensitivity of 0.809 and a specificity of 0.511. Conclusion The PLR and NLR may be related to carotid inflammation in patients and positively correlated with carotid plaque.


Introduction
Diabetes mellitus is a group of metabolic diseases caused by multiple etiologies, clinically characterized by chronic pathological changes in blood vessels and nerves. 1 Diabetic patients constantly suffer from chronic inflammatory reactions, 2 so they are at two-to-four times higher risk of developing a cerebral infarction than non-diabetic patients. 3 Previous research 4 shows that diabetes is an inflammatory disease, and atherosclerosis is the pathological basis of diabetic macroangiopathy. Chronic inflammation is the main reason for its occurrence and development. The adhesion of activated neutrophils on the surface of endothelial cells can cause endothelial dysfunction in patients, and simultaneously, pro-inflammatory factors can further aggravate vascular inflammation. Therefore, long-term inflammation will cause microvascular formation and arteriosclerosis in such patients. 5 Platelet-induced inflammatory response plays a vital role in the development of atherosclerosis in diabetic patients. The mitogenic substances and inflammatory mediators released by activated platelets can recruit more platelets and leukocytes to the inflammatory site, which leads to the occurrence of atherosclerosis. 6 The values of platelet-to-lymphocyte ratio (PLR) and neutrophil-lymphocyte ratio (NLR) are easily obtainable, repeatable, and widely used as markers of inflammation. These two are quick and simple parameters for assessing the state of inflammation and are closely related to the severity of atherosclerosis. 7 Some recent studies have shown that NLR can predict carotid artery stenosis and stroke, which emphasizes the role of NLR as an immunosensor during the process of atherosclerosis. This marker is a powerful predictor of the presence and number of carotid atherosclerotic plaques. [6][7][8][9] Platelet-to-lymphocyte ratio may represent the prethrombotic inflammation status in patients with acute ischemic stroke. The increase in PLR value is closely related to the poor prognosis and the size of cerebral infarction volume. 10 It has been reported that PLR and NLR, as new inflammation indicators, are closely related to the arterial stiffening in diabetic retinopathy (NR) and diabetic nephropathy (DN). 11,12 At present, there are few clinical reports on the relationship between PLR, NLR, and carotid atherosclerotic plaques in young patients with newly diagnosed type 2 diabetes.
Therefore, this study aimed to explore the relationship between carotid plaque and values of PLR and NLR in newly diagnosed young type 2 diabetic patients. The cervical vascular ultrasound was used to judge the existence of carotid atherosclerosis, a semi-quantitative method was used to determine the severity of the plaque, and at the same time, routine and related biochemical indicators of blood were determined. 13

Subjects
The clinical records of 268 young non-consecutive patients with newly diagnosed type 2 diabetes and 104 patients with normal arterial intima, admitted to the Diabetic Medical Department of Baoji Municipal Central Hospital from March 2014 to November 2019, were retrospectively collected. All procedures in this study were approved by the ethics committee of Baoji Municipal Central Hospital.
The inclusion criteria included the following conditions: ① random venous blood glucose > 11.1 mmol/L or fasting blood glucose > 7.0 mmo1/L, OGTT 2-h blood glucose >11.1 mmol/L, and with diabetes symptoms, according to the standards of the "Guidelines for the Prevention and Treatment of Type 2 Diabetes in China (2017 Revision)" 14 ; ② within 48 h from admission; ③ without dietary control or oral hypoglycemic drugs; ④ age of onset between 18 and 45 years old; and ⑤ carotid ultrasound examination conducted within 48 h of admission.
The exclusion criteria included ① other types of diabetes (such as gestational diabetes, type 1 diabetes, and other special types of diabetes); ② combined with severe underlying diseases such as severe heart, liver, kidney function damages; ③ with autoimmune diseases, thyroid and parathyroid diseases, malignant tumors, and other endocrine diseases; ④ those receiving glucocorticoid therapy or hormone replacement therapy; ⑤ patients with previous diabetes medical history; and ⑥ incomplete clinical data and previous medical history.

Study group
According to the test results of carotid intima-media thickness, young patients (observation group) newly diagnosed with type 2 diabetes were divided into the intimamedia-thickening group (116 cases) and plaque group (152 cases). The semi-quantitative method 13 was used to assess the severity of plaque to divide the plaque group into grade I plaque group (62 cases), grade II plaque group (47 cases), and grade III plaque group (43 cases). In the control group, 104 young people with normal arterial intima during the same period were recruited. The data of gender, age, and carotid atherosclerosis-related risk factors (such as hypertension, hyperlipidemia, hyperhomocysteinemia, obesity, smoking, drinking, and family history) were collected. The PLR and NLR between the groups were compared.

Calculation of carotid intima-media thickness and grade
According to the literature, 15 the thickness of carotid intimamedia is determined by the standard, which is ① IMT < 1.0 mm is carotid ultrasound negative and ② IMT ≥ 1.0 mm is carotid ultrasound positive, wherein 1.0 mm < IMT ≤ 1.2 mm is intima-media thickening, and IMT ≥ 1.3 mm is plaque formation. The observed group was divided into intimal thickening group (116 cases) and plaque group (152 cases).
The severity of arterial plaque was assessed by the semiquantitative method. 13 The patients in the plaque group were further divided into 62 cases of grade I plaque, 47 cases of grade II plaque, and 43 cases of grade III plaque by the standard that grade I is unilateral plaque ≤ 2.1 mm; grade II is unilateral plaque > 2.1 mm or both sides have plaque, and at least one of them is ≤ 2.1 mm; and grade III is bilateral plaque >2.1 mm. In addition, 104 healthy young people who underwent carotid B-ultrasound examination for normal carotid intima-media thickness during the same period were selected as the control group. The comparison of baseline data between the groups is shown in Table 1.

Diagnostic criteria for related risk factors
Diagnostic criteria for hypertension. The measurements were taken according to the 2010 Chinese guidelines for the prevention and treatment of hypertension by using a mercurial sphygmomanometer. 16 The patients were not taking blood pressure-lowering drugs. Arterial blood pressure was monitored twice, and the arterial systolic blood pressure 140 mmHg and/or the arterial diastolic blood pressure 90 mmHg was diagnosed as hypertension. In the case of previously diagnosed hypertension, the relevant drugs to control blood pressure were continued to be used.
Diagnostic criteria for dyslipidemia. According to the Guidelines for the Prevention and Control of Dyslipidemia in Chinese Adults (2007), 17 it was important that one of the following conditions was satisfied: ① Total cholesterol (TC = .22 mmo1/L), ② TG = 2.26 mmo1/L, ③ HDL = 01.04 mmol/L, and 4 LDL-C > 4.14 mmo1/L, to be diagnosed as dyslipidemia.
Diagnostic criteria for hyperhomocysteinemia. Hyperhomocysteinemia can be diagnosed on the basis of the following criteria: ① blood homocysteine > 15.0 mmol/L; ② homocysteine-lowering drugs were being used; ③ or doctor-diagnosed hyperhomocysteinemia. 18 Smoking. ① Previous or current regular smoking and smoking > 10 cigarettes/d and ② time > 1 year or smoking cessation < 10 years 19 Drinking. Average alcohol consumption every day, average alcohol intake > 50 g/day 20 Obesity. The formula of body mass index (BMI) is the weight (kg) divided by the square of height (m) (BMI = kg/m 2 ), when the BMI ≥ 27.5 kg/m 2 , it is classified as obesity. 21 Statistical processing SPSS 20.0 software was used for statistical analysis. Measurement data were expressed as mean ± standard deviation (x ± s). An independent Student's t-test or chisquare test was used for comparing the differences between the two groups. One-way ANOVA was used for comparing the differences among three or more than three groups, following the LSD method as a post-hoc test. Variables were put into univariate analysis using logistic regression. Pearson's and Spearman's correlations were determined for normally distributed data and non-normally distributed data, respectively. The receiver-operating characteristic curve (ROC) was used to evaluate the diagnostic values of PLR and NLR. p < .05 was considered as a statistical significance.

Logistic regression analysis of related factors of carotid intima-media thickening in the observation group
First, we analyzed the risk factor of type 2 diabetes using the t-test (Table 1), and the factors that showed statistical significance were added into the following logistic regression analysis. The results showed that diabetes, hypertension, hyperlipidemia, smoking, obesity, PLR values, and NLR values were all related to the incidence of carotid atherosclerosis ( Table 2).

Comparison of PLR and NLR values of patients at admission between the intima-media thickening group and plaque group
An independent student's t-test was used to determine statistically significant differences between the intimamedia-thickening and plaque groups. Results showed that the levels of PLR and NLR in the plaque group were higher than those in the intima-media-thickening group (t = 7.189, 5.387, p = .024, .037). The differences were statistically significant (p < .05) and are summarized in Table 3.

Correlation analysis of PLR and NLR values of patients with different plaque grades
We first assessed the risk factors in the grade I plaque group (62 cases), grade II plaque group (47 cases), and grade III plaque group (43 cases) ( Table 4). As showed in Table 4, no difference was examined in fasting blood glucose (FBG), hypertension, LDL-C, smoking, and BMI among the groups. One-way ANOVA was used to analyze the difference among three plaque subgroups. Results showed that the PLR and NLR values of patients in the grade III plaque group were higher than those in the grade II plaque group (p = .030, .033) and grade I plaque group (p = .021, .027). The levels of PLR and NLR in patients with grade II plaque were higher than those in grade I plaque (p = .036, .041), as shown in Table 5. With the increase in the plaque grade, the values of PLR and NLR also increased. Therefore, there was a positive correlation between PLR and NLR values and plaque grade (r = 0.687, p = 0.012) (Figure 1).  (Table 6 and Figure 2).

Discussion
With the increasing complexity of the modern lifestyle, the work pressure in young people is increasing. The trend of diabetes mellitus in youth is gradually emerging due to the disruption of sleep and circadian rhythms. 22 The fluctuating hyperglycemia can increase the risk of carotid atherosclerosis and other complications compared to persistent hyperglycemia, especially in young people. 23,24 The incidence of diabetes combined with carotid atherosclerosis is growing fast. Chronic inflammation plays a decisive role in the development of diabetic vasculopathy. 25 An earlier study showed that diabetes itself is an inflammatory disease, 2 and the occurrence and development of carotid atherosclerosis are also closely related to the vascular inflammatory reactions. The increased incidence of type 2 diabetes mellitus in youth is closely related to genetic factors and environmental factors, such as the family history of diabetes, unhealthy diet, overweight, excessive fat intake, low frequency of physical activity, long sit-in time, alcohol intake, etc. 26 Some studies have pointed out that hyperlipidemia in pre-diabetic patients is an important risk factor after the thickening of carotid artery intima-media. 27 Among young people, IMT thickening is associated with systolic blood pressure, 28 while smoking also exacerbates atherosclerosis. Therefore, the overall risk of stroke and ischemic stroke has significantly increased. 29 A study found that the majority of young type 2 diabetes mellitus patients were obese, 30 and the increased IMT in young adults was associated with obesity. 31 Type 2 diabetes mellitus in youth becomes quickly aggressive after its onset, as the rate of functional impairment in the islet B cells occurs relatively quickly. Therefore, the risk of comorbidities and death is higher. 32  The reported foreign studies indicated that the incidence of carotid plaque in type 2 diabetes mellitus is 64.3%. 33 This study showed that the risk factors for carotid atherosclerosis in young patients with newly diagnosed type 2 diabetes were mainly hypertension, hyperlipidemia, smoking, and obesity. These outcomes were consistent with the results of previous studies.
Some studies have shown that the basic pathophysiological changes in carotid atherosclerosis plaque (CAP) caused by diabetes are arterial vascular injury and following an inflammatory response. 34 Diabetes mellitus is recognized as an important harmful factor in the formation of CAP. 35 Therefore, detecting the body's inflammatory response is of considerable significance to judge the development and prognosis of atherosclerosis. 36,37 Platelet-to-lymphocyte ratio is considered to be a new biomarker that reflects the degree of systemic inflammation. [38][39][40][41] A high platelet count and low lymphocyte count or high PLR value may contribute to the progression of atherosclerosis and could be associated with adverse events of ischemic prognosis of cardiovascular and cerebrovascular diseases, which reflects the high risk of plaque shedding. 10,42,43 NLR is a risk factor for atherosclerotic plaque formation and arterial stenosis. 44,45 Studies have reported that NLR is associated with blood glucose regulation, insulin resistance, diabetes with acute myocardial infarction, DR, and DN. 46,47 The results of this study showed that the PLR value and NLR value of newly diagnosed type 2 diabetes young patients were significantly higher than those in healthy young people. A series of inflammatory responses caused by hyperglycemia leads to abnormalities of vascular endothelium and blood vessels, thereby mediating the abnormal functional metabolism of the smooth muscle cells. 2,35 The inflammatory factors then result in significantly elevated PLR and NLR values. Abnormal immune function can easily lead to vascular plaque formation, while an unstable plaque can lead to cerebral infarction. 47 Logistic regression showed that PLR and NLR values were significantly related to the onset of arterial plaque in newly diagnosed type 2 diabetes patients. The possible mechanisms are as follows. When oxidative stress and inflammation occur, the release of corticosteroids and catecholamines in the plasma increases, leading to bone marrow suppression. The lymphocyte apoptosis can be observed during plaque formation, rupture, and thrombosis. 48 Under the action of atherosclerosis risk factors, granular proteins released by neutrophils cause endothelial dysfunction. The released proteases can also aggravate endothelial erosion and induce monocyte accumulation in  atherosclerotic lesions. This event can stimulate macrophage maturation, and foam cell formation, and promote atherosclerotic plaque formation. 49 The rapid increase in platelets can change the blood flow rate and promote the synthesis of C-reactive protein and fibrinogen. It can also facilitate the endothelial cells and lymphocytes to produce more inflammatory substances, which in turn can enhance the inflammatory response and the progress of atheromatous plaques. 50 This shows that the PLR and NLR values can be used as novel inflammation indicators to assess the severity of plaque. They can clinically assist the early and rapid screening of patients with high-risk carotid atherosclerosis in making timely and effective treatment strategies. In this study, ultrasound Doppler was used to evaluate the thickness of IMT. The results showed that the PLR and NLR values of patients in the grade III plaque group were higher than those in the grade II plaque group and in the grade I plaque group. The level of PLR and NLR in the patients of plaque group II was higher than that in the grade I plaque group. With the increase of plaque grade, PLR and NLR values of patients also increased due to the aggravation of chronic inflammation. Along with the increased release of inflammatory mediators, the platelets were activated. Various enzymes, receptors, and cytokines prompt further platelet activation, 5 which would cause platelets in the blood to rise quickly. 51 On the contrary, some studies have shown that lymphocytes are usually reduced during the pathogenesis of inflammatory reactions, a relatively common manifestation of the body's stress response, usually accompanied by increased corticosteroids, 44 which may be related to the role of lymphocytes in protecting plaque stability. 52 Through direct contact and the secretion of soluble mediators, platelets and lymphocytes interact and form intercellular aggregates. At the same time, platelets can also promote the recruitment of lymphocytes in the injured blood vessel wall. 6 An increase in platelet count reflects a potential inflammatory response. Some inflammatory mediators stimulate megakaryocyte proliferation to cause the associated increase in platelets. In addition, a high platelet count indicates a high tendency to form platelet-rich thrombocytes on atherosclerotic plaques. However, lymphocytes represent the inactive state of the inflammation control process. 53 The decrease in the total number and a relative number of circulating lymphocytes during an ischemic event may be related to the physiological stress that leads to the increase in cortisol and catecholamine levels, causing the lymphocyte redistribution. 54 The results of this study suggest that the PLR value and NLR value are related to the plaque grade, indicating that the change in these values may reflect the severity of carotid plaque. In clinical work, the PLR and NLR values can be used as effective indicators to measure the severity of carotid artery and inflammatory response. It provides evidence for assessing the condition and guiding treatment modality. At the same time, it is of far-reaching significance to conduct personalized out-of-hospital antiplatelet and antiarteriosclerotic drugs therapy based on different PLR values and NLR values.
However, this study has certain limitations. First, due to time, funding, personnel, and other constraints, this study did not follow up on the selected young patients with newly diagnosed type 2 diabetes, making it hard to understand the dynamic evolution between the CAP, and PLR, and NLR values. Therefore, it could not fully evaluate the relationship between PLR and NLR values and carotid atherosclerosis in young people. Second, the blood routine test has more variability in different periods, and it is arbitrary to make a judgment based on the result of only one blood draw. It may be necessary to test the results multiple times and calculate the average value, which may increase the reliability of the results. Lastly, the number of cases included in this study is relatively small.

Conclusion
The PLR and NLR may be related to carotid inflammation in patients and positively correlated with carotid plaque.  However, the relationship and exact mechanism between PLR, NLR values, and intracranial atherosclerosis in young patients with newly diagnosed type 2 diabetes are not yet clear. We suggest large-scale basic and clinical studies to draw a reliable conclusion.

Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding
The author(s) received no financial support for the research, authorship, and/or publication of this article.

Ethical approval
The study procedure conformed to the ethical guidelines of the Declaration of Helsinki, and the approval for the study was obtained from the Medical Ethics Committee of Baoji Municipal Central Hospital. A written informed consent was obtained from patients enrolled.

Informed consent
A written informed consent was obtained from patients to publish this paper.