Autophagosome protects proximal tubular cells from aldosterone-induced senescence through improving oxidative stress.

Aldosterone exerts an enormous function on proximal tubular cells (PTC) senescence, which is a common pathomechanism contributing to renal dysfunction. Numerous studies have shown that oxidative stress is deeply involved in the pathophysiologic processes of chronic kidney diseases. The study aims to investigate whether autophagy could regulate the process of senescence through oxidative stress in PTC both in vivo and ex vivo. Our results suggested that aldosterone treatment increased the senescence and oxidative stress as evidenced by increased percent of SA-ß-Gal positive cells, reactive oxygen species level, expression of NADPH oxidase 4 (NOX4) rather than NOX2, and the up-regulation of p21 in cultured PTC. Furthermore, the alternation of the expression of p62 and LC3-II/LC3-I demonstrated that aldosterone treatment remarkably influenced autophagic flux. NOX4 siRNA treatment or autophagy induction with rapamycin reduced the oxidative stress and senescence in aldosterone-induced PTC. On the contrary, inhibition of autophagy with chloroquine worsened these changes. Similar results were further confirmed in vivo. Our results suggested that autophagy may become a realistic therapeutic strategy against aldosterone-induced PTC injury via improving oxidative stress.

Role of silence information regulator 1 in regulation of lipopolysaccharide-induced secretion of interleukin-6 and tumor necrosis factor-a in activated BV-2 cells / 中华神经医学杂志

Objective To observe the effect oflipopolysaccharide (LPS) on the cell form of BV-2 cells and the expressions of interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-a) so as to detect the role of silence information regulator 1 (SIRT1) in regulation of LPS-induced proinflammatory cytokines production in activated BV-2 cells.Methods BV-2 cells were divided into control group (normal culture medium) and treatment groups; BV-2 cells in the treatment groups were subdivided into LPS treatment groups,Resveratrol+LPS treatment groups and Sirtinol+LPS treatment groups (cultured with different concentrations of LPS,SIRT1 activator Resveratrol or SIRT1 inhibitor Sirtinol,respectively).MTT assay was employed to identify the cell survival after the inducement.Based on the above MTT results,the cells were then grouped into the control group,LPS treatment group,Resveratrol+LPS treatment group and Sirtinol+LPS treatment group having suitable concentrations of LPS,Resveratrol and Sirtinol; then,the levels of IL-6 and TNF-a were measured with enzyme-linked immuno sorbent assay (ELISA) at 12 and 24 h after the inducement; and the expression of SIRT1 at 24 hafter the inducement was detected by Western blotting.Results As compared with those in the control group,the BV-2 cells in the LPS treatment group had increased cell number,hypertrophic cell body,and shorten cell processes.The cell survival rate increased with increased concentrations of LPS.As compared with those in the control group,the levels of IL-6 and TNF-a in LPS treatment group increased and level of SIRT 1 decreased with significant differences (P＜0.05).Significantly increased levels of IL-6and TNF-a and obviously decreased expression of SIRT1 in the Resveratrol+LPS treatment group were noted as compared with those in the LPS treatment group (P＜0.05); conversely,significantly decreased levels of IL-6 and TNF-a and obviously increased expression of SIRT1 in the Sirtinol+LPS treatment group were noted as compared with those in the LPS treatment group (P＜0.05).Conclusion LPS can change the morphology of BV-2 cells,and induce the levels ofproinflammatory cytokines; impairment of SIRT1 may contribute to such progress obviously.

Protection role of silent information regulator 1 in toxicity of activated BV-2 cells to PC12 cells / 中华神经医学杂志

Objective To observe the effects of silent information regulator 1 (SIRT1) on toxicity of activated BV-2 to PC12 cells and the possible mechanisms.Methods BV-2 microglial cells and PC12 cells were routinely cultured in vitro; ELISA was used to measure to the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) stimulated by lipopolysaccharide (LPS,1 μg/mL) in BV-2 cells.MTT assay was employed to identify the cell viability of PC12 cells injured by culture medium of activated BV-2 and determined the suitable concentrations of resveratrol (a potent SIRT1 activator,5,10,25,50 and 100 μmol/L) and nicotinamide (a known SIRT1 inhibitor,5,10,25 and 50 mmol/L).PC12 cells were divided into groups as follows:control group Ⅱ,LPS+BV-2 co-cultured group,resveratrol treatment group and nicotinamide treatment group (pretreated with resveratrol or nicotinamide for 2 h,and then subjected to culture medium of activated BV-2 cells,in the presence of resveratrol or sirtinol for 18 h); the cell viability was measured by OD value in MTT assay,and the expressions of SIRT1 and acetyl-p53 were detected by Western blotting.Results TNF-α and IL-6 secretions increased gradually at 6,12 and 24 h after LPS being induced BV-2,with significant difference between each two time points (P＜0.05).PC12 cell viability decreased in the LPS+BV-2 co-cultured group as compared with that in the control group Ⅰ,LPS treatment group and BV-2 supernate group (P＜0.05).The cell viability of cells in the 100 μmol/L resveratrol treatment group and 50 μmol/L niacinamide treatment group decreased as compared with that in the control group Ⅱ (P＜0.05),therefore,50 μmol/L resveratrol and 25 μmol/L nicotinamide were chosen in the next experiment.As compared with those in the control group Ⅲ,the cell viability and SIRT1 expression significantly decreased,and acetyl-p53expression significantly increased in the LPS+BV-2 co-cultured group (P＜0.05); as compared with those in the in the LPS+BV-2 co-cultured group,the cell viability and SIRT1 expression significantly increased,and acetyl-p53 expression significantly decreased in the 50 μmol/L resveratrol treatment group,and opposite results were noted in the 25 μmol/L nicotinamide treatment group (P＜0.05).Conclusion SIRT1 can inhibit toxicity of activated BV-2 to PC12 cells,the mechanism of which is partly via p53 activation.

Protective function of human urinary kallidinogenase in rats after focal cerebral ischemic reperfusion and its effect on caspase-3 expression / 中华神经医学杂志

Objective To study the effect of human urinary kallidinogenase (HUK) on neural cell apoptosis in rats after focal cerebral ischemia-reperfusion (FCIR) injury and on Caspase-3 expression.Methods Sixty-six SD rats were randomly divided into sham-operated group (n=6),ischemic-reperfusion group and HUK treatment group.The latter 2 groups were subdivided into 6,12,24,72 and 168 h reperfusion groups (n=6).Middle cerebral artery occlusion models of transient focal cerebral ischemia in the latter 2 groups were established by suture-occluded method. Rats of the HUK treatment group were given tail vein injection of HUK once daily at dosage of 17.5 ×10-3 PNAU/mL and at 1.0 mL/kg manner 3 h after reperfusion. The numbers of apoptotic cells and Caspase-3 positive cells in the cerebral cortex were evaluated with terminal dUTP nick end labeling (TUNEL) assay and immunohistochemistry. Results Cell apoptosis was noted 6 h after the focal cerebral ischemia-reperfusion,reaching its peak level at 24 h,and the apoptotic cells could still be seen at 168 h after the injury.And the expression of Caspase-3 positive cells peaked at 24 h after the injury,and high expression was still noted at 168 h after the injury. The levels of apoptotic cells and the expression of Caspase-3 positive cells in HUK treatment group at different time points (except for 168 h subgroup) decreased significantly as compared with those in ischemic-reperfusion group (P＜0.05). Conclusion HUK may decrease the number of apoptotic cells in the initial 72 h of FCIR injury by down-regulating the Caspase-3 expression.

Differential generation of hydrogen peroxide upon exposure to zinc and cadmium in the hyperaccumulating plant species (Sedum alfredii Hance) / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Sedum alfredii Hance has been identified as zinc (Zn) and cadmium (Cd) co-hyperaccumulator. In this paper the relationships of Zn or Cd hyperaccumulation to the generation and the role of H2O2 in Sedum alfredii H. were examined. The results show that Zn and Cd contents in the shoots of Sedum alfredii H. treated with 1000 micromol/L Zn2+ and/or 200 micromol/L Cd2+ increased linearly within 15 d. Contents of total S, glutathione (GSH) and H2O2 in shoots also increased within 15 d, and then decreased. Total S and GSH contents in shoots were higher under Cd2+ treatment than under Zn2+ treatment. However, reverse trends of H2O2 content in shoots were obtained, in which much higher H2O2 content was observed in Zn2+-treated shoots than in Cd2+-treated shoots. Similarly, the microscopic imaging of H2O2 accumulation in leaves using H2O2 probe technique showed that much higher H2O2 accumulation was observed in the Zn2+-treated leaf than in the Cd2+-treated one. These results suggest that there are different responses in the generation of H2O2 upon exposure to Zn2+ and Cd2+ for the hyperaccumulator Sedum alfredii H. And this is the first report that the generation of H2O2 may play an important role in Zn hyperaccumulation in the leaves. Our results also imply that GSH may play an important role in the detoxification of dissociated Zn/Cd and the generation of H2O2.

Effects of human urinary kallikrein on cell apoptosis of rats with acute focal cerebral ischemia-reperfusion injury / 中华神经医学杂志

Objective To study the effects of human uriilary kallikrein(HUK)on the number of apoptotic cells and the expressions of Bcl-2 and Bax proteins in rats after focal cerebral ischemia and reperfusion(FCIR) injury. Methods Eighty-four Spmque-Dawley(SD)male rats were randomly divided into sham-operated group(n=12),ischemia-reperfusion group(n=36),and HUK-treated group (n=36). Transient focal cerebml ischemia models were established by middle cerebml artery occlusion.Six rats were chosen from sham-operated group,ischemia-reperfusion group,and HUK-treated group for measuring infarct sizes.The rest were used to evaluate neurologic fhnction impaiment and measure the nunlber of apoptotic cells and Bcl-2 or BaX protein positive cells in cerebral cortex with TUNEL and immunohistochemistry.The latter 2 groups were subdivided into 6,12,24,72,168 h reperfusio groups (each n=6). Results The neurologic function impairmlent score,the infarct sizes,the apoptotic cells and the expression of Bax protein of HUK-treated group at different time points (except 168 h group)significantly decreased compared wilh those of ischemia-reperfsion group (p＜0.05).The expression of Bcl-2 protein of HUK-treated group at different time points(except 168 h group) remarkably increased compared with that of ischemia-reperfusion group(P＜0.05). Conclusions HUK can excrt a protection against FCIR injury, maybe through up-regulating Bcl-2 and down-regulating Bax protein in the initial 3 d of FCIR injury to decrease the number of apoptotic cells

Zinc adsorption and desorption characteristics in root cell wall involving zinc hyperaccumulation in Sedum alfredii Hance / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Radiotracer techniques were employed to characterize (65)Zn adsorption and desorption in root-cell-wall of hyperaccumulating ecotype (HE) and non-hyperaccumulating ecotype (NHE) species of Sedum alfredii Hance. The results indicated that at the end of a 30 min short time radioisotope loading period, comparable amounts of (65)Zn were accumulated in the roots of the two ecotypes Sedum alfredii, whereas 2.1-fold more (65)Zn remains in NHE root after 45-min desorption. At the end of 60 min uptake period, no difference of (65)Zn accumulation was observed in undesorbed root-cell-wall of Sedum alfredii. However, 3.0-fold more (65)Zn accumulated in desorbed root-cell-wall of NHE. Zn(2+) binding in root-cell-wall preparations of NHE was greater than that in HE under high Zn(2+) concentration. All these results suggested that root-cell-wall of the two ecotypes Sedum alfredii had the same ability to adsorb Zn(2+), whereas the desorption characteristics were different, and with most of (65)Zn binding on root of HE being available for loading into the xylem, as a result, more (65)Zn was translocated to the shoot.