| Objective:1.In order to explore the influence of 25-(OH)D3 on MCP-1 expression from patients with lupus nephritis(LN),we detected the level of active vitamin D,the expression of MCP-1 m RNA and urinary MCP-1 levels for 24 hours in patients with systemic lupus erythematosus(SLE),and analyzed the correlation between them.2.In order to explore the possible mechanism of the pathogenesis of LN's disease,and to provide a theoretical basis for LN immune regulation treatment,the present study was undertaken on human renal mesangial cells(HRMCs)to preliminarily investigate the effects of lipopolysaccharide(LPS),anti-ds DNA antibody and 1?,25-dihydroxyvitamin D3(1?,25-(OH)2D3)on expression of monocyte chemotactic protein-1(MCP-1).Methods:1.The level of serum 25-(OH)D3,m RNA expression of MCP-1 and urinary MCP-1 levels for 24 hours in 80 LN patients,73 non-LN(n LN)patients and 30 healthy individuals were detected by electrochemiluminescence immunoassay(ECLIA),real time quantitative PCR(RT-q PCR)and enzyme-linked immunosorbent assay(ELISA)respectively.We also analyzed the correlation between serum 25-(OH)D3 level,expression of MCP-1 m RNA and 24-hours urinary MCP-1 levels.2.HRMCs were cultured in vitro and inoculated in 6 well plates.When the cells were grown to about 80%,incubated with serum-free medium at 37? in 5% CO2 for 6 hours to synchronize of cultured cells and then subjected to the following experiments.(1)HRMCs were induced by LPS(0.1?g/ml)with or without four different dosage of 1?,25-(OH)2D3(10-7mol/L,10-8mol/L,10-9mol/L and 10-10mol/L)for 24 hours and 1?,25-(OH)2D3(10-7mol/L)for different time(3h,6h,12 h,24h,48h).(2)HRMCs were treated with three different isotypes of monoclonal anti-ds DNA antibodies(163p.64,163 p.77 and 452 s.160,10?g/ml)for 24 h,48h and 72 h.(3)HRMCs were treated with four different dosage of 163 p.77(1?g/ml,5?g/ml,10?g/ml,15?g/ml)for 72 h.(4)HRMCs were induced by 163 p.77(10?g/ml)with or without four different dosage of 1?,25-(OH)2D3(10-7mol/L,10-8mol/L,10-9mol/L and 10-10mol/L)for 24 hours and 1?,25-(OH)2D3(10-7mol/L)for different time(24h,48 h,72h).The expression level of MCP-1 in the culture supernatant and the expression of MCP-1 m RNA in the cells were detected by ELISA and RT-q PCR respectively.Results:1.(1)The serum 25-(OH)D3 levels:The serum 25-(OH)D3 levels in LN group was significantly lower than the control group and n LN group(p<0.01),and the ratio of the serum levels of vitamin D deficiency in LN group(71.2%)was significantly higher than the control group(23.3%)and n LN group(41.3%)(X2=28.444,p<0.01);(2)The expression of MCP-1m RNA: the expression of MCP-1m RNA of PBMC in n LN group and LN group were higher than control group(p<0.05);(3)The 24 h urinary MCP-1 levels:The 24 h urinary MCP-1 levels in LN group was higher than the control group and n LN group(p<0.05,p< 0.01);(4)The correlation analysis:serum 25-(OH)D3 levels and expression of MCP-1m RNA in n LN group was negatively correlated(r=-0.365,p<0.01),and only in LN group serum 25-(OH)D3 levels and 24 h urinary MCP-1 levels were negatively correlated(r=-0.437,p<0.01).2.(1)1?,25-(OH)2D3 inhibits LPS-induced the expression of MCP-1 in HRMCs:LPS could significantly increase the expression of MCP-1 levels in culture supernatant and MCP-1 m RNA in HRMCs(p<0.01).The expression of MCP-1 in LPS-stimulated HRMCs could supressed by 1?,25-(OH)2D3(p<0.01),especially in the high concentration group and treated for 24 hours.(2)Anti-ds DNA antibody stimulates the expression of MCP-1 in HRMCs:The expression of MCP-1 could be inhanced by stimulating of 163 p.74,163 p.77 and 452 s.160(p<0.01),and the effection was most significantly increased in 163 p.77 treated for 72 h.(3)Different concentrations of anti-ds DNA antibody(163p.77)on expression of MCP-1 in HRMCs:163p.77 10?g/ml and 15?g/ml stimulated for 72 h can significantly increase the expression of MCP-1(p<0.01).(4)1?,25-(OH)2D3 inhibits anti-ds DNA antibody-induced the expression of MCP-1 in HRMCs: 1?,25-(OH)2D3 could significantly reduce the expression of MCP-1 after 163 p.77 intervention(p<0.05),and the inhibitory intensity increased in a certain range with the increase of the concentration and time.Conclusions:1.The deficiency of serum 25-(OH)D3 level and up-regualted urinary MCP-1 level in patients suggested that MCP-1 may be associated with SLE renal injury,and vitamin D may play a role in regulating the expression of MCP-1 of LN.2.1?,25-(OH)2D3 can significantly inhibit the expression of MCP-1 gene and protein after LPS stimulation in HRMCs.The inhibition intensity increased in a certain range with the increase of the concentration of drug and time.3.Anti-ds DNA antibody can promote the expression of MCP-1 gene and protein in HRMCs,and can be inhibited by 1?,25-(OH)2D3.The inhibition intensity increased in a certain range with the increase of the concentration and time.4.1?,25-(OH)2D3 can down-regulate the expression of MCP-1 gene and protein and plays an important role in anti-inflammatory. |
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