中性粒細胞及血小板與淋巴細胞比值與2型糖尿病視網膜病變相關性

吳佩佩，魯 曉，吳清靜，劉彩霞，陳云飛，謝 青

(作者單位:1570100中國海南省?？谑兄心洗髮W湘雅醫學院附屬?？卺t院海南省?？谑腥嗣襻t院眼科;2418000中國湖南省懷化市第一人民醫院眼科)

INTRODUCTION

Diabetic retinopathy (DR) is a common severe microvascular complication of diabetes mellitus(DM)and is the most easily neglected disease among working－age people.According to statistics，China had about 98 million diabetic patients in 2013，making it the country with the largest number of diabetic patients in the world［1］，among which the prevalence rate of DR was 24.7%－37.5%［2］.The pathogenesis of DR is complex and has not been fully elucidated.However，in recent years，it has been found that inflammation has a considerable impact on the occurrence and development of DR［3］.Neutrophil/lymphocyte ratio and platelet/lymphocyte ratio are emerging inflammatory markers in recent years，which have important predictive value for assessing the severity of acute coronary syndrome［4－5］and the prognosis of various malignant tumors［6－7］.For that reason，numerous literatures have reported their relationship with diabetic retinopathy［8－10］.The purpose of this study was to explore the relationship between NLR and PLR and diabetic retinopathy by analyzing and comparing the hematological parameters of type 2 DM.

SUBJECTS AND METHODS

Clinical Data

Inclusion criteria1)Type 2 diabetes is in line with the diagnostic criteria established by WHO in 1999;2)Diabetic retinopathy meets the diagnostic and staging criteria set by the Ophthalmology Group of the Ophthalmological Society of the Chinese Medical Association in 2014;3)Healthy subjects undergoing physical examination at the same time.

Exclusion criteria1)Acute complications of diabetes;2)Acute and chronic infection of the whole body;3)Combined with other eye diseases.

Packet data1)Totally 200 patients with T2 DM were randomly selected from January 2016 to December 2017 in Affiliated Haikou HospitalofXiangyaMedicalCollege，Central South University，and all underwent fundus examination，including fundoscopy，fundus photography or fundus angiography without mydriasis.The patients were divided into DR group(100 cases)that was further divided into non－proliferative retinopathy(NPDR)group(62 cases)and proliferative retinopathy(PDR)group(38 cases)，and DM group(100 cases);2)At the same time，100 healthy subjects in our hospital were selected as normal control group(NCG).We obtained informed consent from all subjects.The study protocol was approved by the Ethics Committee of Affiliated Haikou HospitalofXiangyaMedicalCollege，Central South University.

Methods

General index collectionAll participants were inquired about their medical histories， and their ages， genders，courses of diabetes and systemic disease were recorded，followed by the measurements of systolic blood pressure(SBP)and diastolic blood pressure(DBP)was at rest.

Blood index determinationAll subjects were fasted for 12h，and the elbow vein blood samples were collected afterward in the early morning.The fasting plasma glucose(FPG)，creatinine(Cr)，blood urea nitrogen(BUN)，total cholesterol(TC)，triglycerides(TG)，and high－density lipoprotein(HDL)were measured by automatic biochemical analyzer(Uni Cel DxC 800，Beckman Coulter，USA).Highdensity lipoprotein(HDL)，low－density lipoprotein(LDL)，and glycosylated hemoglobin A1c(HbA1c)was measured by a glycosylated hemoglobin analyzer(Piemier Hb9210，Trinity Biotech，Ireland).Besides，No.XT － 2000i from Sysmex CompanyofJapan was applied notonly to measure neutrophils， lymphocytes， platelets (PLT)， butalso calculate neutrophil count/lymphocyte count(NLR)and platelet count/lymphocyte count(PLR).

Statistical processingIn this study，SPSS 23.0 statistical software was used to analyze the data，and Shapiro－Wilk test was used to evaluate the normality.The measurements of normal distribution were expressed as mean±SD deviation.After logarithmic transformation，the non－normal distribution data were still expressed as median and quartile spacing.In addition，several tests and analyses were used in this study，including Levene test，Variance analysis for comparison between groups which conformed to normal distribution and homogeneity of variance，LSD test for comparison between two groups，Kruskal－Wallis test for comparison between groups which did not conform to normal distribution and heterogeneity of variance，Chi－square test for comparison between the two groups，and Logistic regression analysis for analyzing the risk factors of retinopathy.Enumeration data were expressed by rate，and the significant level was taken by α =0.05 and P＜0.05，indicating a statistically significant difference.

RESULTS

Comparison of General Information in Each GroupThere was no significant difference in sex，age，SBP，DBP among each group(P＞0.05).The duration of DM of NPDR group was significantly longer than that of DM group(P＜0.05).There was no significant difference in the duration of DM between DM group and PDR group，NPDR group and PDR group(P＞0.05).The results are shown in Table 1.

Comparison of Biochemical Indicators in Each GroupThe BUN，TG，LDL，FPG and HbA1c were significantly lower in NCG group than in other groups(P＜0.05).The BUN of DM group was significantly lower than that of PDR group(P＜0.05).The Cr of NCG group was significantly higher than that of DM and NPDR group(P＜0.05).The HDL was significantly higher in NCG group than in other groups(P＜0.05).There was no significant difference in TC among each group(P＞0.05).The results are shown in Table 2.

Comparison of Peripheral Blood Cell Indices in Each GroupOverall，there was no significant difference in the lymphocytes value among each group.However，the PLT value of the control group and PDR group was significantly higher thanthat of DM group and NPDR group(P＜0.05).Furthermore，both neutrophils value and NLR value in PDR group were considerably higher than those in control group，DM group and NPDR group(P＜0.05)，although the NLR value in NPDR group was higher than that in DM group，the difference was not statistically significant(P＞0.05).The PLR value of the control group and PDR group was significantly higher than that of DM group and NPDR group(P＜0.05)，and the PLR value in NPDR group was higher than that in DM group，but the difference was not statistically significant(P＞0.05).The results are shown in Table 3.

Table 1 Comparison of general information in each group

Table 2 Comparison of biochemical indicators in each group

Table 3 Comparison of peripheral blood cell indices in each group

Table 4 Logistic regression analysis

Logistic Regression Analysis of Risk Factors for DR in Patients With and Without DiabetesAll patients were grouped according to whether they had DR or not.The binary Logistic regression analysis was performed with gender，age，course of disease，SBP，DBP，BUN，Cr，TC，TG，HDL，LDL，FPG，HbA1c，Neutrophils，Lymphocytes，PLT，NLR and PLR as covariates.The results showed that the regression coefficients of age，course of disease and SBP were －0.047，0.071 and 0.071，respectively，and the P values were 0.017，0.013 and 0.042 respectively，which were all less than 0.05，

Table 5 Logistic regression analysis

NLR:Neutrophil－to－lymphocyte ratio;PLR:Platelte－to－lymphocyte ratio.indicating statisticalsignificance.Accordingly， ageisa protective factor for DR，while duration and SBP are risk factors for DR patients(Table 4).

Logistic Analysis Using the DR as the Dependent Variable and the NLR，PLR as the Independent VariablesA Logistic regression analysis was performed，using DR as the dependent variable and the NLR，PLR as the independent variable，to determine the association of NLR，PLR with DR.We could observe DR showed a significant association with NLR(P＜0.05)，while DR showed no significant association with PLR(P＞0.05)(Table 5).

DISCUSSION

DR is one of the most common severe microangiopathy in DM，whose occurrence and development involves a great number of pathogenic factors，but the specific pathogenesis has not been fully clarified.Recent studies have shown that diabetic retinopathy is closely related to inflammation，since the expression of inflammatory cytokines such as IL－6［11－13］，IL－1beta［14］and TNF－alpha［15－17］in the vitreous fluid and serum of patients with diabetic retinopathy is significantly with the correlated severity of the disease.Joussen et al［18］identified an important role for TNF－alpha in the pathogenesis of signature diabetic retinopathy pathologies and demonstrated that etanercept(TNF－alpha antagonist)can inhibit diabetic rat retinal cell death.Wu et al［19］found that inocycline decreased the exprssion of IL－1beta，which could protect the retinal vessels.As a result，these studies suggest that inflammation has a significant influence on the pathogenesis of DR.

White blood cell counts and their subtypes are typical indicators of inflammation.Neutrophils play an important role in non－specific immunity，as they can secrete a variety of inflammatory mediators.Lymphocytes are mainly involved in specific immune responses.Platelets are derived from megakaryocytes. Besides participating in the pathophysiological processes of coagulation and thrombosis，activated platelets can release a large number of fine particles.Cytokines and chemokines regulate lymphocyte function，participate in inflammatory reaction and produce immune effects.Inflammatory reactions can increase the number of neutrophils，megakaryocytes and platelets，while the number of lymphocytes decreases.However，the absolute value of the above indicators is susceptible to physiological，pathological and physical environment changes，and their stability is poor.NLR and PLR are new and comprehensive inflammatory markers found in recent years，which can objectively reflect the imbalance of the proportion of cells in the body，and have high stability.Compared with other inflammatory markers，their detection methods are fast，simple and cheap.

In this study，the NLR value of PDR group was significantly higher than that of DM group and NPDR group(P＜0.05)，indicating that the inflammation of PDR group was more serious than that of DM group and NPDR group.At the same time，the NLR value of group NPDR was higher than that of group DM，but the difference was not statistically significant.The PLR value of PDR group was significantly higher than that of DM group and NPDR group(P＜0.05)，which also revealed a more severe inflammation of PDR group than that of DM group and NPDR group.Moreover，the results showed that the order of PLR size was PDR group＞NPDR group＞DM group，but no significant difference was obtained between the two groups.The NLR and PLR values of patients in group PDR were remarkably higher，suggesting that the occurrence and development of DR are closely related to inflammation.

The Logistic regression analysis of risk factors for DR in diabetic patients demonstrated that age was a protective factor for DR，duration of DM and SBP were risk factors for DR.Consistent with our research，Wong et al［20］reported the prevalence and risk factors of diabetic retinopathy in a multiethnic cohort in the US，which demonstrated that the age was a protective factor for DR.A study in USA［21］aimed at adults with diabetes aged 40 years and older showed that the longer duration of DM was an independent risk factor for DR.The UKPDS［22］reported that the higher systolic blood pressure was independently associated with diabetic retinopathy.

On the contrary，the Logistic regression analysis of risk factors for DR in diabetic patients demonstrated that no significant difference was found between NLR and PLR，indicating that NLR and PLR were not independent risk factors for DR in DM patients.Meanwhile， aLogistic regression analysiswas performed，using DR as the dependent variable and the NLR，PLR as the independent variable，to determine the association of NLR，PLR with DR.We could observe DR showed a significant association with NLR(P＜0.05)，while DR showed no significant association with PLR(P＞0.05)，suggesting that the occurrence and development of DR are closely related to NLR.Yue et al［8］reported that the NLR and PLR ratios in DR group were significantly higher than those in DR group.Ulu et al［9］announced that the NLR values were closely related to the severity of DR，while Oztürk et al［23］considered the NLR values to be associated with diabetic microvascular complications and suggested that they could be used for early diagnosis of diabetic microangiopathy.Huang et al［24］used logistic regression analysis to show that NLR was a risk factor for predicting DN and DR in type 2 diabetic patients.They also used ROC analysis to evaluate the predictive accuracy of NLR and other models to predict DR risk in patients.The auROC of the NLR was 0.669(95%CI:0.606－0.732).In the same dataset，WBCs had an auROC of 0.576(95%CI:0.512－0.641)，a neutrophils of 0.635(95%CI:0.573－0.698)，a lymphocytes of 0.585(95%CI:0.520－0.651)，a creatinine of 0.555(95%CI:0.491－0.619)，significantly lower than that of the NLR(all P＜0.001).In the present study，DM patientshavenotyetunified hypoglycemic regimen，and there are considerable differences among longterm use of different drugs，different patients with compliance，diagnosis as well as treatment process.From a clinical point of view，they may be confounding factors，thus affecting NLR and PLR values，which can be further explored by a large sample correlation study of multiple factors.Considering that this study is cross－sectional and that neutrophils，lymphocytes and platelets have a short life span and a large number of fluctuations，reflecting only a specific time point，rather than a long－term state of the body，the impact of NLR and PLR on DR may not be sufficient to offset the impact of other risk factors at the current time point.

To sum up，NLR and PLR values in the PDR group increased，but the risk factors for DR did not.This study is a small sample of retrospective analysis，which still need to be further explored for the clinical treatment of DR to provide new ideas.