Serum levels of complement B and D in patients with diabetic peripheral neuropathy and their correlation / 中国医师杂志

Objective:To investigate the serum level and significance of complement factor B (CFB) and complement factor D (CFD) in patients with type 2 diabetes mellitus (T2DM) and diabetic peripheral neuropathy (DPN).Methods:From October 2019 to October 2020, 110 patients with T2DM in the endocrinology department of Affiliated Hospital of Jining Medical College were divided into DPN group ( n=60) and simple T2DM group ( n=50) according to whether or not DPN was combined. In addition, 52 cases of physical examination population in the physical examination center in the same period were selected as the normal control group ( n=52). The serum levels of CFB, CFD and tumor necrosis factor-α (TNF-α) were measured by enzyme linked immunosorbent assay (ELISA). The correlation between CFB, CFD and clinical indexes was analyzed, and the influencing factors of DPN were analyzed by logistic regression. Results:The serum levels of CFB and CFD in DPN group were higher than those in T2DM group and normal control group [CFB: (845.43±101.10)μg/ml vs (792.19±116.59)μg/ml, (739.20±123.43)μg/ml, P<0.05], [CFD: (491.71±41.03)mg/L vs (467.58±45.16)mg/L, (445.16±50.47)mg/L, P<0. 05]. Pearson correlation analysis showed that the serum level of CFB was positively correlated with glycosylated hemoglobin (HbA 1c), fasting plasma glucose (FPG) and TNF-α (all P<0.05) and negatively correlated with triiodothyronine (FT3) and total bilirubin (TBIL) (all P<0.05). Serum CFD level was positively correlated with systolic blood pressure, HbA 1c, FPG and TNF-α (all P<0.05), but negatively correlated with FT3 and TBIL (all P<0.05). Logistic regression analysis showed that CFB and CFD were still influential factors for the occurrence and development of DPN after excluding confounding factors such as systolic blood pressure, HbA 1c, FPG, FT3, DBIL, TBIL and TNF-α. Conclusions:(1) Serum CFB and CFD levels were significantly increased in DPN patients, suggesting that CFB and CFD may be involved in the occurrence and development of DPN. (2) Serum TNF-α level was significantly increased in DPN patients, confirming the role of TNF-α in the pathogenesis of DPN.

The change of serum complement factor B and its clinical implication in children with primary IgA nephropathy / 中华实用儿科临床杂志

Objective:To detect serum level of complement factor B (CFB), and to explore its correlations with clinical parameters and prognosis in children with primary IgA nephropathy (IgAN).Methods:A total of 204 children with primary IgAN confirmed by kidney biopsy in the Department of Nephrology of the First Affiliated Hospital of Zhengzhou University from December 2014 to April 2017 were included in IgAN group.During the same period, 84 healthy children were included in healthy control group.Their mean age was (11.0±3.5) years and (10.9±3.2) years, respectively.Patients in IgAN group were divided into low CFB group (102 cases) and high CFB group (102 cases) according to the medium serum level of CFB measured by enzyme-linked immunosorbent assay. Spearman′ s coefficient was employed to analyze correlation amongst various parameters.Multivariable-adjusted Cox proportional ha-zards models were used to evaluate the relationship between serum CFB level and prognosis in children with IgAN. Results:Serum CFB levels were significantly higher in IgAN group than that in healthy control group [290.9 (186.2-453.9) mg/L vs.218.9 (155.0-321.3) mg/L, Z=-3.372, P=0.001]. Serum levels of CFB were negatively correlated with serum albumin ( r=-0.388, P<0.001) and estimated glomerular filtration rate ( r=-0.416, P<0.001), but positively correlated with serum creatinine ( r=0.305, P<0.001) and 24 h urinary protein ( r=0.456, P<0.001) in IgAN group.The incidences of crescents (C1-2) (70.6% vs.29.4%, χ2=34.588, P<0.001) and C 3 deposition (+ + -+ + + ) (63.7% vs.44.1%, χ2=7.892, P=0.005) were significantly higher in high CFB group than those in low CFB group. Kaplan- Meier analysis showed that high CFB levels predicted worse renal outcome in pediatric IgAN patients ( χ2=17.509, P<0.001). Multivariate Cox regression analysis showed that the high CFB level was the independent risk factor for the poor renal outcome ( HR=2.517, 95% CI: 1.284-4.932, P=0.007). Conclusions:High serum levels of CFB are associated with decreased renal function, increased urinary protein excretion, crescentic formation and poor renal outcome in pediatric IgAN patients.

Construction and assessment of recombinant plasmid pRNAT-U6.1/CFB siRNA and its inhibitory effect on proliferation of human umbilical vein endothelial cells / 中华实验眼科杂志

Background Choroidal neovascularization (CNV) is one of the causes of blindness in multiple eye diseases.Researches showed that complement system participates in the pathogenesis of CNV.Objective This study was to construct the recombinant of complement factor B-small interference RNA (CFB-siRNA) expression vector and to observe its inhibitory effect on human umbilical vein endothelial cells (ECV-304).Methods CFB gene primers were designed based on human CFB gene,and an expression vector of CFB-siRNA was constructed by inserting CFB-siRNA into pRNAT-U6.1/Neo plasmid.Recombinant plasmids were confirmed by the digestion analysis of restriction endonuclease,and all inserted sequences were verified by DNA sequencing.The recombinant pRNAT-U6.1/CFB-siRNA plasmid and the blank plasmid were transfected into ECV-304 cells in the CFB-siRNA group and blank plasmid group by electroblot,respectively,and non-transfected cells served as the normal control group.The cells were observed under the fluorescence microscope 48 hours after transfection,and the transfective efficiency was calculated.The relative expression of CFB mRNA in the cells of different groups was detected by semi-quantitative reverse transcription PCR (RT-PCR).MTT was employed to calculated the growth inhibitory rates of the cells 24,48 and 72 hours after transfection.The percentages of the cells in different cell cycles were detected by flow cytometry.Results The sequence of the target vector was identical to the designed sequence.The green fluorescence protein (GFP) was seen in both the CFB-siRNA group and the blank plasmid group.The relative expression levels of CFB mRNA were 0.07 ±0.04,0.14 ±0.02 and 0.14 ±0.03 in the CFB-siRNA group,the blank plasmid group and the normal control group,respectively,a significant difference was obtained among the three groups (F=233.05,P =0.00);the expression level of CFB mRNA in the CFB-siRNA group was significantly declined in comparison with the blank plasmid group and the normal control group (both at P＜0.05).The growth inhibitory rates of the cells were (23.45 ±0.01) ％,(33.48 ±0.02) ％ and (45.49±0.01) ％ at 24,48 and 72 hours after transfection,respectively,a significant difference was obtained among the three groups (Fgroup =212.99,P =0.00);the growth inhibitory rates in CFB-siRNA group were significantly higher than that in the blank plasmid group and normal control group (all at P＜ 0.05).The percentages of G1 phase cells were (44.4 ±0.5) ％,(25.8 ±0.4) ％ and (27.9 ± 0.6) ％ in the CFB-siRNA group,the blank plasmid group and the normal control group respectively,a significant difference was obtained among the three groups (F=58.98,P=0.00).The percentages of G1 phase and G2 phase cells in the CFB-siRNA group were significantly higher than those in the blank plasmid group and the normal control group (all at P＜0.05).Conclusions Recombinant pRNAT-U6.1/CFB siRNA inhibits the proliferation of ECV-304 cells effectively by arresting the cells in G1 intermediate phase of the growth cycle.

The role and significance of serum B factor in the pathogenesis of systemic lupus erythematosus / 中国医师杂志

Objective To investigate the expression of serum B factor in the peripheral blood of patients with systemic lupus erythematosus (SLE),and explore its role in the pathogenesis.Methods Seventy eight patients with SLE in our hospital and 46 healthy persons were eligible to participate in this study.Rate nphelometyr was used to test serum B factor for 78 patients with SLE and 46 healthy controls.According to systemic lupus erythematosus disease activity index (SLEDAI),participants were divided into steady SLE group (SLEDAI ＜ 5) and active SLE group (SLEDAI ≥5),which was further divided into mild,moderate,and serious subgroups.The differences in serum B factor between SLE patients and healthy controls,including SLE patients with different severity,were all compared.Then we analyzed the differences in serum B factor and other laboratory and clinical indexes between active and steady SLE patients.The correction of serum B factor and other laboratory and clinical indexes were also analyzed.Results Compared to healthy controls,patients with SLE had significantly lower value of serum B factor [(27.13 ± 8.98) mg/dl vs (36.73 ± 5.47) mg/dl,t =7.4,P ＜ 0.01].Compared to steady SLE group,SLE active group had significantly lower level of serum factor B,C3 and C4,and also had significant higher level of C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR) (all P ＜ 0.05).Moreover,There were significant differences in the lower level of serum B factor between subgroups.Correlation analysis showed that the level of serum B factor was negatively associated with the levels of CRP and SLEDAI scores,whereas serum B factor was positively associated with the levels of C3 and C4 (all P ＜ 0.05).Conclusions Serum B factor is related to SLE.Serum B factor might be involved in the pathogenesis of SLE.Detection of serum B factor is helpful for diagnosis and evaluation of SLE disease activity.

Inhibition efficacy of CFB-siRNA on laser-induced choroidal neovascularization in rat / 中华实验眼科杂志

BackgroundChoriodal neovascularization is an important ocular manifestation of angiogenesis in eyes,which derives from the choroid capillaries.Recent studies have found that complement activation is playing a key role in the laser-induced CNV.Because of the key position of CFB in the alternative pathway,bytargeting CFB and blocking the alternative pathway may provide an approach to observe the role of this alternative pathway in the generation of CNV.Objective This study was to investigate the inhibitory effect of reconstructed complement factor B (CFB)-small interfering ribonucleicacid(siRNA)on choroidal neovascularization (CNV)and its mechanism. Methods Experimental CNV was induced by laser photocoagulation in 96 eyes of 48 clean Brown Norway rats.The rats were randomly divided into 4 groups.25,50 and 75 μg B factor siRNA were injected via caudal vein on 1 day,3,5 days after photocoagulation in different dose groups,and normal saline solution was injected at the same way in experimental control group.Other 12 normal rats were used as blank control group.Fundus fluorescein angiography(FFA) was performed on 3,7,14,21,28 days after injection of CFB-siRNA and CNV was scored.The expressions of vascular endothelial growth factor(VEGF) and factor Ⅷ in choroid were detected by immunochemistry.The expressions of CFB-siRNA,VEGF,transforming growth factor β2( TGF-β2 )proteins in choroid were determined using immunochemistry in 7,14,21,28 days,and the expressions of mRNA of CFB-siRNA,VEGF,TGF-β2 were examined by reverse transcription polymerase chain reaction(RT-PCR). ResultsFFA revealed that the CNV rates in various doses of CFB-siRNA groups were significant lower than those of experimental control group in various time points(P＜0.05),and those in 75 μg B factor siRNA were decreased in comparison with 25 μg B factor siRNA (P＜0.05).Immunochemistry showed that the intensities of the VEGF and factor Ⅶ expression in various doses of CFB-siRNA groups were weaker than the blank control group ( P ＜ 0.05 ).Compared with the control group,the expression of CFB reduced in 7 days,and then approached to the level near the control group.Fourteen to twenty-one days after injection of CFB-siRNA,VEGF and TGF-β2 depressions in different doses of CFB-siRNA groups were lower than blank control group( P＜0.05 ).CFB expression in choroid showed the lower levels in CFB-siRNA injection group compared with blank control group in from 7 through 21 days (P＜0.05).RT-PCR displayed the gradual increase of CFB mRNA and curve-like changes of VEGF and TGF-β2 with time prolong. Conclusions Recombinated CFB-siRNA can effectively inhibit laser-induced CNV by down-regulating the expression of VEGF and factor Ⅷ.Alternative pathway of complement plays an important role in the production of CNV.

Alteraciones del sistema complemento en pacientes con anemia drepanocítica en estado basal / Alterations of the complement system in patients with basal sickle cell anemia

Se determinó la actividad de las vías clásica y alternativa del complemento, así como la cuantificación de algunos de sus componentes en 46 pacientes con anemia drepanocítica (AD) (hemoglobina SS), 21 sin complicaciones clínicas y 25 con estas; entre las más frecuentes encontramos: número de infecciones, úlceras maleolares, crisis vasooclusivas, hepáticas y aplásticas, considerando como límite un año anterior a la toma de muestra de sangre. Se demostró una disminución significativa de la actividad de la vía alternativa, factor B y del componente C3 en el grupo de pacientes con complicaciones clínicas. Se observó una correlación significativa entre el número de crisis vasooclusivas e infecciones y la actividad de la vía alternativa, el factor B y el C3. Estos resultados sugieren que los fenómenos inflamatorios que pueden persistir en pacientes con AD en estado basal, fundamentalmente aquellos con historia anterior de complicaciones clínicas, pueden provocar alteraciones en la actividad del complemento.

The activity of the classic and alternative complement pathways, as well as the quantitation of some of their components was determined in 46 patients with sickle cell anemia (haemoglobin SS), 21 with no clinical complications and 25 with them. Among the most frequent complications we found: number of infections, malleolar ulcers, and vaso-occlusive, hepatic and aplastic crises. A year previous to the blood sample taking was considered as the limit. A significant reduction of the activity of the alternative pathway, factor B and component C3 was proved in the group of patients with clinical complications. A marked correlation was observed between the number of vaso-occlusive crises and infections and the activity of the alternative pathway, factor B and complement C3. These results suggest that the inflammatory phenomena that may persist among patients with basal sickle cell anemia, mainly those with prior history of clinical complications, may produce alterations in the complement activity.