Relationship Between Activation And Apoptosis Of PBMC In Patients With IgAN And Triptolide Intervention

IgA nephropathy is the most common form of primary glomerular disease in the developed world, is characterized by macroscopic hematuria and diffuse deposition of IgA in the glomerular mesangium. Immunological cells and cytokines played the central role in the pathogenesis of IgA nephropathy. TNF-a,IL-6 and NO are the particularly interesting molecules in the inflammatory and immune response. Those cytokines are now strongly suspected in the induction or aggravation of renal damage in various glomerulonephritides. More evidence showed that the lymphocytes apoptosis is associated with autoimmune disease. Dates showed that the inhibition of B cell death causes the development of IgA Nephropathy in (New Zealand White×C57BL/6)F1-bcl-2 Transgenic mice. Further studies are needed to determine whether the expression of cytokines and apoptosis abnormality in peripheral blood mononuclear cells (PBMC) play the centrol role in the pathogenesis of IgAN.Tripterygium Wilfordii Hook f has been widely used in the treatment of IgA nephropathy because of its unique anti-inflammatory and immunosuppressive effects. It can relieve the renal lesions and alleviate hematuria. However, the cellular and molecular mechanisms underlying TP-induced immunosuppressive and anti-inflammatory in PBMC from IgAN patients are not fully understood. The purpose of this study is to investigate the expression of TNF-a,IL-6,NO and apoptosis of PBMC from IgAN patients in vitro and explore the possible mechanisms for TP of immunosuppression and apoptotic induction. Objective:To investigate the expression of TNF-a,IL-6,NO in the PBMC from IgAN patients, probe into its correlation with pathogenesis,clinic manifestation and pathological type.Methods:29 patients with IgA nephropathy were enrolled. PBMC were seperated from them and 16 normal controlls. The expressions of TNF-a and IL-6 mRNA in PBMC of IgAN patients (group IgAN) and normal people (group CN) were measured with RT-PCR, and the concentrations of serum TNF-α,IL-6 and NO were measured by ELISA.Results:1. The group IgAN had increased TNF-a and IL-6 mRNA expression by their PBMC compared with group CN (P<0.05; P<0.01);2. Total TNF-a,IL-6 and NO serum levels were clearly elevated in the group IgAN compared with the group CN (P<0.01), total TNF-a,IL-6 and NO were intercorrelated in these patients.3. Serum TNF-a,IL-6 and NO levels obtained from patients with grade III or IV renal histopathological findings were higher than in those with grade I or II histopathology. In addition, the protein concentrations of TNF-a,IL-6 in serum obtained from patients with renal dysfunction (Ccr<60ml/mim) were markedly higher than those with normal renal function(Ccr>60ml/mim), the serum NO was lower than those with normal renal function.4. A positive correlation was noted between the serum TNF-a level and quantity of protein excretion in the urine (r=0.27, P<0.05), whereas histopathological findings correlated with serum TNF-a,IL-6 levels(r=0.56, P<0.01; r=0.38, P<0.05; r=0.31,P<0.05). However, there was no significant correlation between serum TNF-a,IL-6 concentrations and hematuria or the serum IgA,C3 levels.Conclusion:Our data suggest that the expression and production of TNF-a,IL-6 and NO were increased in IgAN, play a role in disease progression, and be one target of immunosuppressive therapy. Objective:To investigate the role of apoptosis of the PBMC and proteins modulation in the pathogenesis of IgAN.Methods:The early apoptosis rate of PBMC of IgAN patients and healthy controls were measured by AnnexinⅤ-FITC staining method and flow cytometry. The expression of Bcl-2,Bax proteins were detected by Western Blot. All the data were analyzed by SPSS 13.0 statistics, software.Results:1. The early apoptosis rate of PBMC of IgAN patients was higher than that of the healthy controls (P<0.01). In addition, the apoptotic rate in IgAN patients with more than 1.0 g/day proteinuria were higher than those with less than 1.0 g/day proteinuria. There was a significant positive correlation between the apoptotic rate of IgAN and the serum levels of TNF-α,IL-6 (TNF-a/apoptotic rate:t=2.79, P<0.05; IL-6/ apoptotic rate:t=2.77, P<0.05)2. Expression of Bcl-2 protein was increased in IgAN patients compared with healthy controls, but the statistic difference is not significant (P=0.09)3. The expression of Bax protein was not differ between the IgAN patients and healthy controls.Conclusion:Accelerated apoptosis of PBMC in IgAN may participate in IgAN pathogenesis. Increased expression of the suppressive apoptotic protein Bcl-2 could result in the inappropriate longevity of autoimmune lymphocytes, which may result in excessive production of IgA. Objective:To investigate the immune inhibition by TP on PBMC of IgAN patients.Methods:The cytotocity of TP was detected by indirect method by MTT. The TNF-a and IL-6 mRNA levels were tested by Realtime-PCR, and culture supernatant of PBMC which had been exposed to different level doses for a certain time was collected, and the the content of TNF-a and IL-6 was measured by ELISA, and the concentration of nitric oxide was detected by Griess reagent. The early apoptosis rates of TP on PBMC from IgAN patients were measured by AnnexinⅤ-FITC staining method and flow cytometry, the expressions of Bcl-2,Bax,Caspase 9,Caspase 3 mRNA and proteins were detected by RT-PCR and Western Blot.Results:1. The cell viability in the presence of TP decreased in a dose and time-dependent manner.2. TP (12.5ng/ml,25ng/ml) could significantly inhibit the expression of TNF-a,IL-6 mRNA and protein. The secretion of NO was also markedly surpressed by TP.3. TP(12.5ng/ml,25ng/ml) induced a progressive increase apoptosis of PBMC. The expression of Bcl-2 mRNA and protein was down-regulated, meanwhile the levels of Bax,Caspase 9,Caspase 3 mRNA and protein expression were up-regulated.Conclusion:TP had an inhibitory effect on the inflammation-promoting cytokines of TNF-a,IL-6 and NO. TP induces apoptosis of PBMC from IgAN patients, and this effect may at least in part through the mitochondrial pathway. 1. The expression and production of proinflammatory molecules TNF-α,IL-6,NO were increased in IgAN, play a role in disease progression, and be one target of immunosuppressive therapy.2. Accelerated apoptosis of PBMC in IgAN may participate in IgAN pathogenesis. Increased expression of the suppressive apoptosis gene protein Bcl-2 could result in the inappropriate longevity of autoimmune lymphocytes, which may result in excessive production of IgA.3. TP had an inhibitory effect on the inflammation-promoting cytokines of TNF-α,IL-6 and NO. TP induces apoptosis of PBMC from IgAN patients, and this effect may at least in part through the mitochondrial pathway.