| Objective:Systemic lupus erythematosus（SLE）is an autoimmune disease with many factors and the appearance of multiple autoantibodies,which often affects multiple systems and organs.The research of domestic and foreign scholars have shown that regardless of the length of time that SLE is diagnosed,cardiovascular disease（CVD）is always the leading cause of death in SLE patients,and SLE patients have a higher risk of CVD,which mainly related to the acceleration of atherosclerosis（AS）.Previous studies have found that the imbalance between T helper cells 17（Th17）and regulatory T cells（Tregs）is one of the important reasons for the high incidence of AS in SLE patients.Regulatory B cells（Bregs）can indirectly regulate the balance of Th17/Treg cells and thus inhibit the release of inflammatory cytokines.Therefore,in this study,we compared the expression of Bregs,Th17 and Tregs related cytokines in the serum of SLE patients with early-onset AS,SLE patients,and healthy people.We established LDLr-/-mice+Pristane model,detected the expression of Breg cells in SLE mice with early-onset AS,and its regulation of Th17/Treg balance and its influence on the release of downstream inflammatory factors through RT-PCR,flow cytometry,ELISA and other experiments,and analysed its possible molecular mechanisms,providing theoretical basis for the prevention and targeted treatment of SLE with early AS.Methods:1、Patients diagnosed with SLE admitted to the Department of Rheumatology and Immunology of Affiliated Hospital of Guilin Medical College from April 2020 to January 2021 were collected.According to the color Doppler ultrasound examination of the carotid artery,the SLE patients were divided into groups.SLE patients with carotid intima-media thickness（CIMT）≥1.0mm were selected as the SLE-AS group.SLE patients with CIMT<1.0mm were included as the SLE-non AS group.Cytokines（IL-2,IL-4,IL-6,IL-10,IL-17,IFN-γ,TNF-α）test results,general clinical data（such as gender,age）,course of disease,Body Mass Index（BMI）,SLE disease activity index（SLEDAI）,globulin,blood lipid combination（TG,CHO,HDL,LDL,APO-A,APO-B,LPa）,immune group（Ig M,Ig A,Ig G）,complement（C3,C4）,C-reactive protein（CRP）,erythrocyte sedimentation rate（ESR）,24-hour urine protein level in SLE patients were collected.Healthy subjects matched with SLE patients in gender and age at the Health Examination Center of the Affiliated Hospital of Guilin Medical College during the same period were selected as the control group.The test results of cytokines（IL-2,IL-4,IL-6,IL-10,IL-17,IFN-γ,TNF-α）and general clinical data（such as gender and age）were collected for statistical analysis.2、The model of SLE with early-onset AS was constructed by intraperitoneal injection of Pristine reagent into 10 8-week-old LDLr-/-mice as the SLE-AS group,and 10 MRL/lpr mice of the same age as the SLE group,10 C57BL/6 mice served as the control group,all were females.After 14 weeks of high-fat feed,the mice were weighed,and the skin,hair,lymph nodes and spleen were recorded and observed.After removing the eyeballs and taking blood,test the levels of albumin,blood lipids（TG,CHO,LDL,HDL）,complement C3,and immunoglobulin（Ig G）.The serum was taken to detect antinuclear antibodies（ANA）,anti-double-stranded DNA（ds DNA）antibodies,cytokines（IL-10,IL-17,IL-35,TGF-β,TNF-α）by enzyme-linked immunosorbent assay（ELISA）.The kidney tissue was taken for HE staining and PAS staining,and the aorta was taken for oil red O staining.After taking the spleen,the expression of Bregs,Th17 cells,and Tregs was detected by flow cytometry,and the expression of IL-10,RORγt,and Foxp3 m RNA were detected by RT-PCR.Results:1.The results of the clinical study:（1）A total of 29 patients in the SLE-AS group and 106 patients in the SLE-non AS group were enrolled.None of them had a history of smoking and were treated with corticosteroids.The control group included 242 people.（2）The age,duration of disease,BMI,CHO,LDL,APO-B,LPa and ESR levels of the SLE-AS group were higher than those of the SLE-non AS group,and the difference was statistically significant（P<0.05）.（3）Compared with the control group,the serum levels of IL-2,IL-6,IL-10,IFN-γand TNF-αin the SLE-non AS group were significantly increased,and the serum IL-6 level in the SLE-AS group was increased,the difference was statistically significant（P<0.05）.However,there was no significant difference in cytokine levels between SLE-AS group and SLE-non AS group（P>0.05）.（4）Multivariate logistic regression analysis showed that age（OR 1.081,95%CI 1.025,1.141;P=0.004）and BMI（OR 1.402,95%CI 1.134,1.732;P=0.002）were risk factors for SLE with AS.（5）In the SLE-AS group,serum IL-2 was negatively correlated with disease activity SLEDAI（r=-0.517）,IL-6 was positively correlated with globulin（r=0.388）and IFN-γwas positively correlated with Ig M（r=0.537）,all of which were statistically significant（P<0.05）.（6）The ratio of serum IL-10/IL-17 in SLE-AS group was negatively correlated with TG,CHO,LDL,APO-B and24-hour urine protein,with statistical significance（P<0.05）.2.The results of basic research:（1）LDLr-/-mice suffered from intraperitoneal injection of Pristine reagent and fed with high-fat diet,and the symptoms of hair loss became more obvious with the increase in age.MRL/lpr lupus mice also appeared hair loss performance,normal C57BL/6 mice do not have the above phenomenon.（2）After 14 weeks of high-fat diet,there was no significant difference in body weight of mice in the LDLr-/-+Pristane group（SLE-AS group）,MRL/lpr+normal saline group（SLE group）and C57BL/6+normal saline group（Control group）（P>0.05）,but the serum albumin of mice in the SLE-AS group and the SLE group was significantly lower than that of the Control group（P<0.05）.（3）The serum TG,CHO and LDL levels of mice in the SLE-AS group were higher than those in the SLE group and Control group,while the level of HDL was lower,and the difference was statistically significant（P<0.05）.（4）Compared with Control group,SLE-AS group and SLE group showed a decreased complement C3 level and an increased Ig G level,with statistical significance（P<0.05）.（5）Mice in SLE-AS group and SLE group had significantly increased serum ANA and anti-ds-DNA antibody levels（P<0.05）and pathologically showed kidney disease.Oil-red O staining of aorta in SLE-AS group showed lipid deposition in orange,but no lipid deposition in SLE group or Control group.（6）The IL-35levels of SLE-AS group and SLE group were lower than those of the Control group,while the levels of TNF-αand IL-10 were higher than those of the Control group.The difference was statistically significant（P<0.05）.Compared with mice in the Control group,the levels of IL-17 and TGF-βin the SLE group increased,and the difference was statistically significant（P<0.05）.The serum IL-10 level of SLE-AS group mice was significantly higher than that of SLE group mice,which was statistically significant（P<0.05）,but there was no significant difference in the levels of IL-17,IL-35,TGF-βand TNF-αbetween the two groups（P>0.05）.（7）Compared with mice in the Control group,the proportion of Treg cells in the spleen lymphocytes of the SLE-AS group and SLE group was significantly reduced,while the proportion of IL-10+Breg and Th17cells was significantly higher.The difference was statistically significant（P<0.05）.There was no significant difference in the ratio of Treg and Th17 cells in the spleen lymphocytes of the SLE-AS group and the SLE group（P>0.05）.The ratios of Breg cells and Th17/Treg cells in SLE-AS group and SLE group were negatively correlated with statistical significance（P<0.05）.（8）Compared with the Control group,the expression levels of Foxp3 m RNA in the SLE-AS group and the SLE group were significantly reduced,while the expression of RORγt m RNA were significantly increased,and the difference was statistically significant（P<0.05）.There was no significant difference in Foxp3 and RORγt m RNA expression between SLE-AS group and SLE group（P>0.05）.The expression of IL-10 m RNA in SLE-AS group was significantly higher than that of SLE group and Control group,with statistical significance（P<0.05）.Conclusions:（1）The age of SLE patients with AS in this study was（49.17±8.52）years,suggesting that they have an early onset trend.The occurrence of AS in SLE patients may be closely related to the levels of BMI,CHO,LDL,APO-B,LPa,and ESR.（2）The age,course of disease,BMI,CHO,LDL,APO-B,LPa,ESR levels of SLE patients with AS were significantly different from those of SLE patients without AS.Among them,age and BMI were risk factors for SLE with AS.（3）The level of serum inflammatory cytokines in SLE patients is significantly higher than that of normal healthy people.Some inflammatory cytokines in SLE patients with AS were related to immune progression;the ratio of IL-10/IL-17may be closely related to dyslipidemia and renal disease in SLE patients with AS.（4）LDLr-/-mice were successfully induced by intraperitoneal injection of Pristane in a mouse model of SLE with early-onset AS,showing similar phenotypic characteristics,serological changes,immunological abnormalities,and renal lesions as human SLE patients.LDLr-/-+Pristane mice can have obvious hyperlipidemia and atherosclerotic plaque lesions when fed with high-fat diet.（5）There were obvious inflammatory responses and imbalance between Breg/Th17/Treg cells in SLE mice with early-onset of AS.（6）Breg cells may regulate the imbalance of Th17/Treg cells mainly through the secretion of IL-10. |
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