Sublyticc5b-9 Induction Of Irf-1 Expression Of Thy-1 Nephritis Rat Gmcs Apoptosis Lesions

Part I The role of IRF-1 in the rat GMCs apoptosis induced by sublytic C5b-9Objective: To investigate the effects of sublytic C5b-9 complexes on the production of interferon regulatory factor 1 (IRF-1), apoptosis of glomerular mesangial cells (GMCs) and Caspase activation. Then to explore the role of IRF-1 on the mechanism of sublytic C5b-9 mediating GMCs apoptosis and production of actived Caspase in vitro. Methods: Firstly, the rat GMCs were cultured and divided into different groups. The mRNA and protein levels of IRF-1 in GMCs with sublytic C5b-9 stimulation were measured by RT-PCR and Western blot. The GMCs apoptosis was evaluated by flow cytometry analysis. Meanwhile, the protein levels of cleaved Caspase 9,cleaved Caspase 8 and cleaved Caspase 3 were measured by Western blot. Moreover, the effects of IRF-1 overexpression by IRF-1 expression plasmid or knockdown by IRF-1 short hairpin RNA (shRNA) on the GMCs apoptosis were determined using the corresponding methods, and the production of actived Caspase induced by sublytic C5b-9 in IRF-1 knockdown GMCs were measured by Western blot. In addition, the number of GMCs apoptosis induced by sublytic C5b-9 or overexpression of IRF-1 were also measured when GMCs were pretreated with Caspase 8 inhibitor (Z-IETD-FMK) and Caspase 3 inhibitor (Z-DEVD-FMK). Results: In vitro, the IRF-1 expression, GMCs apoptosis and Caspase activation induced by sublytic C5b-9 could be increased, and IRF-1 can promote GMCs apoptosis induced by sublytic C5b-9. The number of GMCs apoptosis and the production of cleaved Caspase 8 and cleaved Caspase 3 protein were markedly reduced by IRF-1 knockdown. In addition, the number of GMCs apoptosis induced by sublytic C5b-9 or overexpression of IRF-1 could be markedly reduced by Caspase 8 inhibitor (Z-IETD-FMK) and Caspase 3 inhibitor (Z-DEVD-FMK). Conclusion: GMCs apoptosis mediated by sublytic C5b-9 in vitro is partially dependent on the up-regulation of IRF-1 expression and Caspase activation.PartⅡThe inhibitory effect of silencing IRF-1 gene on the GMCs apoptosis and secondary proliferation of the rat with Thy-1 nephritis[Abstract] Objective: To explore the inhibitory effects of silencing renal IRF-1 gene using shRNA on the GMCs apoptosis and secondary proliferation of rats with Thy-1 nephritis (Thy-1N). Methods: Firstly, an animal model of rat Thy-1N was induced by injecting anti-thymocyte serum (ATS). Then the expression of IRF-1 and actived Caspase in the renal tissue were observed at fixed time. Furthermore, rats were divided into different groups, and IRF-1 shRNA expressing plasmids, Caspase 8 inhibitor (Z-IETD-FMK) and Caspase 3 inhibitor (Z-DEVD-FMK) were transferred into different rat kidneys by renal artery perfusion followed by electroporation or by tail vein injection, and then Thy-1N was reproduced by injection of ATS. The protein levels of IRF-1 and cleaved Caspase 9, 8 and 3 in the renal tissue of different group rats were assessed by Western blot at 3h after ATS administration. Meanwhile, the renal histopathologic changes were observed by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), electron microscopy (EM) and light microscope (LM). The total content of 24h urinary protein was also detected at 24h and 7d. Results: The expression of IRF-1 in the renal tissue was obviously increased at 3h in Thy-1N rats, and the expression of IRF-1 and cleaved Caspase 8 and 3 in the renal tissue transferred with IRF-1 shRNA followed by induction of Thy-1N was significantly reduced compared with Thy-1N model rats. TUNEL analysis and morphological observation under EM showed that the GMCs apoptosis in IRF-1 shRNA+Thy-1N group, Z-IETD-FMK+Thy-1N group and Z-DEVD-FMK+Thy-1N group were markedly reduced compared with Thy-1N model group. The secretion of 24h urinary protein in IRF-1 shRNA+Thy-1N group, Z-IETD-FMK+Thy-1N group and Z-DEVD-FMK+Thy-1N group were also less than that in Thy-1N model group or DMSO+Thy-1N group at 7d, and morphological observation under LM showed that the total number of glomerular cells in IRF-1 shRNA+Thy-1N group, Z-IETD-FMK+Thy-1N group and Z-DEVD-FMK+Thy-1N group were significantly decreased compared with Thy-1N model group. Conclusion: Silencing renal IRF-1 gene or inhibiting the Caspase activation can diminish the lessions of GMCs apoptosis including secondary proliferation of rats with Thy-1N.Part III The role and signal pathway of triggering IRF-1 transcription or expression by sublytic C5b-9[Abstract] Objective: To study the function of sublytic C5b-9 in IRF-1 transcription and to explore the probable signal pathway involved in it. Methods: Firstly, the 1300bp, 600bp, 200bp and 100bp fragments of rat IRF-1 promoter were amplified by PCR with pGL3-Basic/IRF-1 promoter 1700bp as a template and were directionally cloned into pGL3-Basic multiple cloning sites to construct the luciferase reporter plasmids, namely, pGL3-Basic/IRF-1 promoter 1300bp, pGL3- Basic/IRF-1 promoter 600bp, pGL3-Basic/IRF-1 promoter 200bp and pGL3-Basic/IRF-1 promoter 100bp. After being screened and comfirmed, the recombinant plasmids and pGL3-Basic/3×GAS were transfected into GMCs with PRL-SV40 respectively. Then the rat GMCs were divided into different groups, and calculate the relative luciferase activity unit (RLU) of each group. Secondly, the rat GMCs were cultured and divided into different groups, and the protein levels of signal transducers and activators of transcription (STAT), p-STAT1, p38, p-p38, c-Jun N-terminal kinase (JNK), p-JNK, extra cellular signal-regulated protein kinase (ERK) and p-ERK in GMCs with sublytic C5b-9 stimulation were measured by Western blot. The protein levels of p-STAT1 and IRF-1 in GMCs stimulated by sublytic C5b-9 in the present or absent of JAK inhibitor (AG490) and the protein levels of IRF-1 in GMCs stimulated by sublytic C5b-9 in the present or absent of p38 inhibitor (SB203580), JNK inhibitor (SP600125) and ERK inhibitor (U0126) were also detected by Western blot. At last, the protein levels of p-STAT1 in GMCs stimulated by sublytic C5b-9 in the present or absent of p38 inhibitor (SB203580) were measured by Western blot. Results: The luciferase analysis verified the IFN-gamma activated sequence (GAS) plays a major role in the induction of IRF-1 by sublytic C5b-9 attack, and the expression of p-STAT1 and IRF-1 can be downregulated using the JAK and p38 inhibitor. Conclusion: Sublytic C5b-9 can upregulation IRF-1 promoter activity in rat GMCs, which is relevant with the activation of p38MAPK and JAK-STAT pathways.