Sublytic Of C5b-9 Induced Gmcs Proliferation And Secretion Of Ecm Molecular Mechanisms Of Its Signaling Pathway

Partâ… The role of sublytic C5b-9-induced synthesis ofTsp-1,TGF-β₁ and proliferation of glomerular mesangial cells(GMCs) including secretion of extracelluar matrix (ECM)AIMS: To investigate the synthesis of thrombospondin-1 (Tsp-1) andtransforming growth factor-β₁ (TGF-β₁) in sublytic C5b-9-stimulatedglomerular mesangial cells (GMCs) and explore the effects of sublyticC5b-9 complex on the proliferation of GMCs and secretion of extracelluarmatrix (ECM) in vitro, furthermore, to detect the relationship betweenTsp-1, TGF-β₁ and the proliferation of GMCs, the secretion of ECM.METHODS: The GMCs were divided into different groups. The level ofmRNA and protein of Tsp-1, TGF-β₁ cell cycle protein (cyclin D₂),fibronectin (FN) were measured by real-time polymerase chain reaction(PCR). Meanwhile, the content of activated TGF-β₁ in cultured supernatantwas detected by ELISA. The proliferation of GMCs was evaluated by³H-thymidine incorporation (³H-TdR). In addition, the blocking peptide ofTsp-1 (GGWSHW, inhibit the activation of TGF-β₁) was utilized to studythe function of Tsp-1 on the sublytic C5b-9-induced the synthesis ofTGF-β₁, proliferation of GMCs and secretion of ECM. RESULTS: Thelevel of mRNA and protein of Tsp-1, TGF-β₁ induced by sublytic C5b-9(5% anti-Thy 1 antibody and 4% normal serum) was up-regulated at 18 h,and the addition of sublytic C5b-9 to GMCs apparently increased theexpression of cyclin D₂, FN compared with other groups at 24 h. At sametime, sublytic C5b-9-induced GMCs were sufficient to augment the number of cells. The blocking peptides of Tsp-1 can reduce sublytic C5b-9-inducedcontent of TGF-β₁ and inhibit the mRNA and protein increase of FN, yethas no effect on the proliferation of GMCs. CONCLUSIONS: Theseresults indicate that sublytic C5b-9 can induce the expression of Tsp-1,TGF-β₁ and promote rat GMCs proliferation as well as ECM secretion.Tsp-1 and TGF-β₁ may play a role in the secretion of ECM. Partâ…¡The effect of silencing Pi3-k/Akt gene on sublyticC5b-9-induced the synthesis of Tsp-1, TGF-β₁ andproliferation of glomerular mesangial cells (GMCs) includingsecretion of extracelluar matrix (ECM)AIMS: To explore the effect of silencing phosphatidylinositol 3-kinase(Pi3-k)/Akt signal pathway on the synthesis of sublytic C5b-9-inducedTsp-1, TGF-β₁ and proliferation of GMCs, secretion of ECM from thelevel of gene. METHODS: Eight pairs of small interference RNAs (siRNA)were designed against different regions of Pi3-k/Akt target genes. RatGMCs in vitro were transfected with all eight small hairpin RNAs (shRNA)and optimized Pi3-k shRNA (shPi3-k) and Akt shRNA (shAkt) respectively,then divided into different administration groups. The expression of Tsp-1,TGF-β₁, cyclin D₂ and FN mRNA was assessed by Real-time PCR and theprotein level of phosphorylated Akt (P^*-Akt), total-Akt (T-Akt), Tsp-1,cyclin D₂ and FN was determined by Western blotting. The content ofTGF-β₁ in cultured supernatant was detected by ELISA. The proliferationof GMCs was evaluated by ³H-TdR. RESULTS: In rat GMCs transfectedwith Pi3-k and Akt shRNA expression vectors, shPik3c3-2 and shAkt1-4could markedly reduce the expression of P^*-Akt and T-Akt respectively.The GMCs transfected with shPik3c3-2, shAkt1-4, shPik3c3-2+shAkt1-4,then stimulated with sublytic C5b-9, the expressions of Tsp-1, TGF-β₁cyclin D₂ and FN as well as the number of GMCs were all obviouslydecreased. The decrease mentioned above was most obvious in cellstransfected with shPik3c3-2 or shPik3c3-2+shAkt1-4. CONCLUSIONS: Sublytic C5b-9 can induce the synthesis of Tsp-1, TGF-β₁ and promoteproliferation of GMCs, secretion of ECM via Pi3-k/Akt signal pathway.