Study On The Protective Effect Of Polydatin On Hypoxia/Re-oxygenation Induced Human Renal Proximal Tubular Epithelial Cell Injury

PartⅠTHE MECHANISM OF SIGNAL TRANSDUCTION PATHWAY HYPOXIA/RE-OXYGENATION INDUCED HUMAN RENAL PROXIMAL TUBULAR EPITHELIAL CELL INJURYObjective: To investigate the mechanism of signal transduction pathway in hypoxia/re-oxygenation (H/R)-induced human renal proximal tubular epithelial cell line (HK-2 cell) injury in vitro.Methods: The HK-2 cells were divided into 3 groups: the control group, the H/R group and the Pyrrolidine dithiocarbamate (PDTC) treatment group. The HK-2 cells were grown in DMEM with normoxic condition in control group. Hypoxic culture conditions were produced by placing the cells in a mini-incubator filled with 95%N2/ 5%CO2 gas mixture saturated with DMEM (PH7.4). Re-oxygenation was performed by placing the cells in an incubator with 95% air/5% CO2 gasses. The HK-2 cells in H/R group were exposed to hypoxia for 24 hours and re-oxygenation for 24 hours. The HK-2 cells with PDTC were cultured in hypoxia condition for 24 hours, and then cultured in normoxic condition for 24 hours in PDTC treatment group. Temperature was maintained at 37oC. The samples were obtained and tested when hypoxia for 6h, 12 h, 24 h, and then re-oxygenation for 6h,12h and 24h. The HK-2 cell's quantity was assessed by MTT assay. The protein expressions of nuclear factor-κB (NF-κB) protein65 were measured by immunohistochemistry. The ICAM-1mRNA expressions were detected by RT-PCR and the ICAM-1 protein, the enzyme-linked immunosorbent assay (ELISA).Results:The number of HK-2 cells were significantly decreased after exposure to hypoxia for 6 hours, continuously decreasing to the lowest level at 24h and increased slightly after re-oxygenation in H/R group, with significant statistical differences between the H/R group and the control group at any corresponding time points(P<0.05). The expression of HK-2 cell NF-κB protein65 (average optical), the level of ICAM-1 mRNA transcription (average optical ratio) and the ICAM-1protein expression (pg/ml) were significantly increased in H/R group compared with those in control group at any corresponding time point (P <0.05). The number of HK-2 cells were increased, but the expressions of HK-2 cell NF-κB protein65, ICAM-1mRNA transcription and ICAM-1 protein in PDTC group were decreased, compared with those in control group or H/R group at any corresponding time points,with statistical significance(P <0.05).There existed a significant positive correlation between the expression of NF-κB protein65 and ICAM-1 protein expression since the Pearson correlation coefficient was r=0.821, P <0.050.Conclusions:1. The hypoxia/re-oxygenation induced HK-2 cell injury model was successfully duplicated by putting the HK-2 cells in serum-free and glucose-free DMEM and a tank filled with 95%N2/5%CO2 to simulate the ischemia-reperfusion conditions in vivo. The method is simple, easy and with stable results.2 Hypoxia/re-oxygenation culture conditions may induce HK-2 cell NF-κB activation, stimulate the ICAM-1mRNA transcription and ICAM-1 protein expression in a time dependent manner. PDTC treatment could produce a significant decrease H/R HK-2 cell ICAM-1mRNA transcription and ICAM-1 protein expression.3. The mechanism of intercellular signal transduction during H/R induced HK-2 cell injury maybe initiated the activation of NF-κB, then increased the expressions of ICAM-1 on the surface of HK-2 and finally promoted adhesion of PMNs. PartⅡSTUDY ON THE PROTECTIVE EFFECT OF POLYDATIN ON HYPOXIA/RE-OXYGENATION INDUCED HUMAN RENAL PROXIMAL TUBULAR EPITHELIAL CELL INJURYObjective: To study the protective effect of polydatin on hypoxia/re-oxygenation induced HK-2 cell injury in vitro.Method: The experimental HK-2 cells were divided into 4 groups: the control group, the H/R group, the PDTC treatment group and the PD treatment group. The HK-2 cells were grown in DMEM with normoxic condition in control group. Hypoxic culture conditions were produced by placing the cells in a mini-incubator filled with 95%N2/ 5%CO2 gas mixture saturated with DMEM (PH7.4). Re-oxygenation was performed by placing the cells in an incubator with 95% air/5% CO2 gasses. The HK-2 cells in H/R group were exposed to hypoxia for 24 hours and re-oxygenation for 6 hours. The HK-2 cells with PDTC were cultured in hypoxia condition for 24 hours, and then cultured in normoxic condition for 24 hours in PDTC treatment group. Temperature was maintained at 37oC. The samples were obtained and tested when hypoxia for 6h, 12 h, 24 h, and then re-oxygenation for 6h,12h and 24h. The HK-2 cell's quantity was assessed by MTT assay. The expressions of NF-κB p65 were measured by immunohistochemistry. The ICAM-1mRNA expressions were detected by RT-PCR and the ICAM-1 protein, the ELISA.Results: The HK-2 survival cells at any corresponding time point were significantly increased in PD treatment group than those in H/R group, with statistical differences (P < 0.05). The HK-2 cells NF-κB p65 expressions, ICAM-1mRNA transcriptions and the ICAM-1 protein expressions at any corresponding time point were decreased in PD treatment group than those in control group, with statistical differences (P<0.05). There were no significant differences in any measured values at any corresponding time point between the PDTC treatment group and the PD treatment group (P>0.05).Conclusion: This data demonstrate that Polydatin may have a protective effect on hypoxia/re-oxygenation induced NK-2 cell injury, presumably via inhibiting the activation of NF-κB, down-regulating the ICAM-1mRNA transcription and ICAM-1 protein expression on the surface of HK-2 and finally reducing PMNs adhesion.