Protective Mechanism Of NALP3-si-RNA On Rat Renal Tubular Epithelial Cells From Hypoxia/Reoxygenation Injury

Objective To investigate the influence of hypoxia/reoxygenation (H/R) on the NALP3expression of rat renal tubular epithelial cells (NRK-52E). And to explore the mechanism ofprotecting cells from hypoxia/reoxygenation injury by constructing specific small interferenceRNA (siRNA) to inhibit NALP3 expression in rat renal tubular epithelial cells (NRK-52E).Methods (1) To establish the H/R injury model of NRK-52E by regulating the pressureof N2 in incubator to hypoxia condition, the cells were cultured with hypoxia for 1h and thenwith reoxygenation for 1h、2h、4h、8h、16h and 24h.The activity of lactae dehydrogenase (LDH)in the culture medium, cell count and cell viability, the expression of NALP3 were determinedby biochemical method, trypan blue exclusion and Western blot. (2) The siRNA was designedand synthesized based on rat NALP3 complete sequence, and was transfected into NRK-52E.Theirrespective siRNA transfected group was used as control.NALP3 expression was examined byWestern blot.(3) The cells were cultivated to 4 groups: control group; H/R group；irrespectivesiRNA transfected group and NALP3 siRNA transfected group. To establish the H/R injurymodel of NRK-52E by regulating the pressure of N2 in incubator to hypoxia condition, the cellswere cultured with hypoxia for 1h and then with reoxygenation for 4h.The models wereevaluated by the activity of lactae dehydrogenase (LDH) in the culture medium. And theexpression of NALP3 was determined by Western blot. (4)Cellular apoptosis was examined byAnnexin V/PI staining and flow cytometry; NF-κB DNA binding activity, IκB-α, Bcl-2 and Baxexpress was examined by EMSA and Western blot.Results (1)Compared with the control group, the activity of LDH significantlyincreased, cell count and cell viability significantly decreased（all p﹤0.05). The expression ofNALP3 significantly increased and reached to peak at 4h after H/R. (2) The specific siRNAcould efficiently inhibit NALP3 expression in NRK-52E. Compared with the irrespective siRNAtransfected group, the protein expression of NALP3 was significantly down-regulated in NALP3siRNA transfected group (all p﹤0.05).(3) After hypoxia 1h and reoxygenation 4h, the activity ofLDH and the expression of NALP3 increased. Compared with the irrespective siRNA transfected group, LDH concentration in media and the expression of NALP3 significantly decreased inNALP3-siRNA transfected group. (4) After hypoxia 1h and reoxygenation 4h, the activity ofLDH increased. NF-κB DNA binding activity was increased, IκB-αphosphorylation anddegradation, Bcl-2 and Bax were significantly up-regulated. However, compared with theirrespective siRNA transfected group , NF-κB DNA binding activity、IκB-αdegradation andBax/Bcl-2were significantly decreased (p﹤0.05) in NALP3 siRNA transfected group. At thesame time, the ratio of apoptosis was significantly increased in the three groups than in control,compared with the irrespective siRNA transfected group, the ratio of apoptosis was significantlydecreased (p﹤0.05).Conclusions H/R induces the expressions of NALP3 in NRK-52E. The synthe sizedsiRNA can inhibit the express of NALP3 and protect NRK-52E from hypoxia/reoxygenationinjury. The mechanism might be via desuppressing the activation of NF-κB; modulatingexpression of Bcl-2 and Bax, as well as decreasing cell apoptosis.