| Objective: Currently, the incidence of kidney disease, especially diabetic nephropathy(DN) is increasing in our country. DN is one of the most serious complications of diabetes. It often leads to late development of terminal renal failure, a serious threat to human public health. The pathogenesis of DN is very complex, involving heredity, metabolism, growth factors and cytokines. With the deepening of the study, it was found cytokines play an important role in the occurrence and development of DN. Tumor necrosis factorα(TNF-α) can not be ignored as a key cellular factor. In DN animal models and patients, the expression of TNF-αwas increasing, suggesting that TNF-αwas involved in the process of DN.TNF-αis composed of 157 amino acids. In addition to hematopoietic cells, renal intrinsic cells, maily including mesangial cells, glomerular cells and tubular epithelial cells, also produce this cytokine. TNF-αhas a wide range of biological activity and participates in cell differentiation, proliferation and apoptosis. It was reported that TNF-αcan mediated apoptosis of opossum kidney proximal tubular epithelial cell. However, the role of TNF-αinducing human renal proximal tubular epithelial cell apoptosis was rarely reported.One of the important cytokine signal transduction pathways is Janus kinase/signal transducers activator of transcription (JAK/STAT) signaling pathway, which closely related to the development of kidney disease. STAT is a JAK kinase substrate and coupling of tyrosine phosphorylation signaling pathways, which play a role in transcriptional regulation. Suppressor of cytokine signaling proteins (SOCS) family is a class of negative regulation factors that can block cytokine signal transduction. SOCS family plays a negative regulatory role in JAK/STAT signaling pathway. SOCS may act a protective effect in DN by inhibiting the JAK/STAT signaling pathway. It was proved that TNF-αinduced apoptosis is closely related to JNK and NF-κB signal transduction pathway. But the relationship among TNF-αinduced apoptosis, SOCS-1and JAK/STAT signaling pathway is barely understood. In this experiment, we mainly observed TNF-αinduced HKC apoptosis and activat JAK/STAT signaling pathway and the effect of JAK/STAT signal specificity blockers (AG490) and SOCS-1 overexpression on TNF-α- induced HKC apoptosis and JAK/STAT signaling pathway. Preliminary discussion the effect of TNF-α, SOCS-1 gene on apoptosis and p-STAT1, p-JAK2 protein expression level in JAK/STAT signaling pathway in the hope of providing a new basis and strategies for DN.Methods :1 Materials: HKC, pCR3.1-SOCS-1 transfected HKC, pCR3.1-vector transfected HKC are permanent cell clons, TNF-α, AG490.2 Methods:2.1 We choose methyl thiazolyl tetrazolium (MTT) to determine proliferation of cell in HKC and to determine the half inhibitory concentration (IC50) of TNF-α: established of the control group (control), solvent control group (solvent) and drug group. Set up the drug concentration: 5 ng.ml-1, 10 ng.ml-1, 20 ng.ml-1, 40 ng.ml-1, 80 ng.ml-1, 160 ng.ml-1. Action time: 24 h, measure IOD values, calculate the rate of cell growth inhibition, and determine IC50 according to the formula.2.2 Experimental groupsThe cells are divided 5 groups: control group (N), TNF-αgroup (T), TNF-α+ AG490 group (T+A), TNF-α+ pCR3.1-SOCS-1 transfection group (T+S1), TNF-α+ pCR3.1-vector transfection group (T+V).2.3 Detecting index2.3.1 Detected the apoptosis in various group cells by flow cytometry (FCM).2.3.2 Apoptosis of various group cells induced by TNF-αis detected by DNA ladder.2.3.3 Detected the apoptosis in various group cells by TUNEL.2.3.4 Western blot was used to detect the expression of p-STAT1 and p-JAK2 proteins in various groups.Results:1 The effect of TNF-αon the proliferation in HKCMTT assay shows: The IC50 TNF-αacting on HKC for 24 h was 18.59 ng.mL-1. TNF-αcan inhibite HKC proliferation, and with the drug concentration increasing, the inhibition effect gradually increased. When the drug concentration reached 160 ng.mL-1, the inhibition rate of this drug is close to 80%. Solvents show no significant effect on cell growth (p>0.05).2 TNF-αinduced HKC apoptosis and the effect of TNF-αon the expression of p-STAT1 and p-JAK2 proteins FCM, DNA Ladder and TUNEL show: Compared with control, afterTNF-αstimulating HKC 6 h, the apoptosis rate increased significantly, reaching a peak at 12 h and then declined, but still higher than control group (p < 0.05).Western blot results show that compared with the control, after TNF-αstimulating HKC, with the stimulation time extened, the expression of p-STAT1 and p-JAK2 protein gradually increased, reaching a peak at 12h and then decline, but still significantly higher than control group (p<0.05). 3 The effect of AG490 on TNF-αinduced HKC apoptosis and the expression of p-STAT1 and p-JAK2 proteinsFCM, DNA Ladder and TUNEL show that compared with T group, the apoptosis rate of T+A group droped significantly (p<0.05), but still higher than control group (p<0.05).Western Blot results show that compared with T group, the expression of p-STAT1 and p-JAK2 proteins of T+A group reduced significantly (p<0.05), but still higher than control group (p<0.05).4 The effect of SOCS-1 overexpression on TNF-αinduced HKC apoptosis and the expression of p-STAT1 and p-JAK2 proteinsFrom the results of FCM, DNA Ladder and TUNEL analysis, compared with T group, the apoptosis rate of T+V group was not obviously different; the apoptosis rate of T+S1 group discreased significantly (p<0.05), but still higher than control group (p<0.05).From the results of Western blot analysis, compared with T group, the expression of p-STAT1 and p-JAK2 proteins of T+S1 group reduced significantly (p<0.05), but still higher than control group (p<0.05). In T + V group, the two kinds of protein expression was not significantly different compared with T group.Conclusions:1 TNF-αcan inhibite HKC proliferation in a dose dependent manner and induce HKC apoptosis. After TNF-αstimulating HKC, p-STAT1 and p-JAK2 protein expression increased. AG490 intervention can inhibit TNF-α-induced HKC apoptosis and p-STAT1 and p-JAK2 protein expression, suggesting that JAK/STAT signaling pathway involved in the process of TNF-αinduced HKC apoptosis.2 Overexpression of SOCS-1 can inhibit TNF-α-induced HKC apoptosis and p-STAT1 and p-JAK2 protein expression, prompting SOCS-1 may inhibite TNF-α-induced HKC apoptosis through the JAK/STAT signaling pathway negative feedback.
|
PDF Full Text Request