| ObjectiveTumor necrosis factor(TNF)-like weak inducer of apoptosis(TWEAK)is a recently identified proinflammatory cytokine of the TNF superfamily which has multiple biological activities and plays an important role in immune inflammatory diseases.Recent work has revealed that TWEAK may play an important role in the pathogenesis of kidney damage,who acting via its sole receptor,fibroblast growth factorinducible 14(Fn14),inducing inflammatory responses,angiogenesis,mesangial proliferation,filtration barrier injuries,renal fibrosis,etc.Therefore,to focus on TWEAK/Fn14 signaling-mediated renal injury may be promising in the treatment of patients with LN.This study was to investigate the levels and clinical significance of urinary tumor necrosis factor-like weak inducer of apoptosis(TWEAK)in childhood-onset systematic lupus erythematosis with lupus nephritis(LN cSLE).To observe the effect of recombinant human tumor necrosis factor-like weak inducer of apoptosis(rhTWEAK)on autophagy and apoptosis when stimulating human embryonic kidney cells 293(HEK293),then exploring the interaction between autophagy and apoptosis to reveal the potential mechanism of TWEAK in LN.Methods(1)Detection of urinary TWEAK levels by ELISA:(1)Participants: A total of 33 cSLE(?18 years old).Among them,3 males and 30 females,aged 3 months to 18 years(median age 12 years old),which including 13 LN cSLE.A total of 30 aSLE patients(> 18 years old,and started after 18 years old).Among them,5 males and 25 females,aged 20 to 63 years old(median age 34 years old),which including 15 LN aSLE.(2)The clinical data and related indicators were collected,which including age,gender,blood routine,urine routine,dsDNA,serum C3,C4,24-hour urine protein,SLEDAI and so on.(3)Urine specimen collection: Collecting 10 ml of morning urine of the subject,centrifuge at low temperature high speed centrifuge,and the supernatant was dispensed and frozen at-80? for testing.(4)Quantitative detection: According to the instructions of the ELISA kit,a standard curve was drawn to quantitatively measure the level of urinary TWEAK.(5)Relevance analysis of disease activity: SPSS software was used to analyze the correlation between urinary TWEAK levels with clinical indicators such as dsDNA,serum C3,C4,24-hour urine protein,and SLEDAI.(2)The effect of rhTWEAK on autophagy in HEK293 cells:(1)Grouping and drug intervention: The HEK293 cells in an exponential growth phase were cultured for 48 h and divided into two groups treated with rhTWEAK: time gradient group(0H,6H,12 H,20H rhTWEAK)and concentration gradient group(0ng/ml,10ng/ml,50ng/ml,100ng/ml rhTWEAK).(2)Western blot and immunofluorescence assay were used to detect the expression level of autophagy regulator LC3 B after time gradient rhTWEAK intervention.(3)Western blot and immunofluorescence assay were used to detect the expression level of autophagy regulator LC3 B after concentration gradient rhTWEAK intervention.(3)The effect of rhTWEAK on apoptosis in HEK293 cells:(1)Grouping and drug intervention: As(2)(1).(2)Apoptosis was identified based on cell morphology by using the viability dye DAPI.Results(1)Urinary TWEAK levels ELISA tests(1)The level of urinary TWEAK in the LN cSLE group(231.951 ± 116.630)was significantly higher than that in the non-LN cSLE group(32.396 ± 26.761)and the normal control group(31.713 ± 21.900)(P<0.001),while there was no significant difference in urinary TWEAK level between the non-LN cSLE group(32.396 ± 26.761)and the normal control group(31.713 ± 21.900)(P>0.05).(2)The level of urine TWEAK in LN aSLE group(236.738 ± 97.253)was significantly higher than that in non-LN aSLE group(39.893±17.955)(P<0.001),while there was no significant difference in urinary TWEAK level between LN cSLE group(231.951 ± 116.630)and LN aSLE group(236.738 ± 97.253)(P>0.05).(3)Correlation analysis showed that the TWEAK level in the LN cSLE group was positively correlated with the SLEDAI score(r=0.65,P<0.05)and the rSLEDAI score(r=0.92,P<0.001),while negatively correlated with serum C3(r =-0.94,P < 0.001)and serum C4(r =-0.85,P < 0.001),and was positively correlated with urine protein(r = 0.64,P < 0.001).(2)The effect of rhTWEAK on autophagy in HEK293 cells Western Blot and immunofluorescence results:(1)Compared with the 0H 100ng/ml rhTWEAK control group,Western Blot showed that the regulatory factor LC3 B protein in the 6H,12 H,20H 100ng/ml rhTWEAK experimental group were distinctly increased(P<0.001,<0.001,<0.001)and were in a time-dependent manner.Similar results were observed by immunofluorescence assay(P<0.001,<0.001,<0.001).(2)Compared with the 0ng/ml rhTWEAK control group,Western Blot showed that the protein LC3 B was significantly increased in the 10 ng/ml,50 ng/ml,100 ng/ml rhTWEAK experimental group(P<0.001,<0.001,<0.001)moreover thoses were in a dose-dependent manner.Similar results were observed by immunofluorescence assay(P<0.001,<0.001,<0.001).(3)The effect of rhTWEAK on apoptosis in HEK293 cells DAPI staining results:(1)Compared with the 0H 100ng/ml rhTWEAK control group,the apoptotic rate of the 6H,12 H,20H 100ng/ml rhTWEAK experimental group were significantly increased(P<0.001)in a time-dependent manner.(2)Compared with the 0ng/ml rhTWEAK control group,the apoptotic rate of the 10 ng/ml,50 ng/ml,and 100 ng/ml rhTWEAK experimental groups were also significantly increased(P<0.001)in a dose-dependent manner.Conclusion(1)Urinany TWEAK test specimens are easy to collect and non-invasive,which is significantly associated with SLE renal injury.The expression level can dynamically reflect the activity of LN disease and is an ideal biomarker for LN cSLE.(2)There is no significant difference in the expression of urine TWEAK between LN cSLE and LN aSLE,and it is unable to distinguish the LN cSLE and LN aSLE.(3)TWEAK participates in the pathogenesis of LN by promoting the occurrence of autophagy and apoptosis in HEK293 cells. |
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