3.1 PMB decreased cells viability and promoted apoptosis in NRK-52E cells in a dose- and time-dependent way.
We initially tested the toxicity of PMB in vitro, NRK-52E cells were treated with different dosages of PMB about 12, 24, 36, 48 h. CCK8 assay showed that PMB treatment decreased viability of NRK-52E cells in a dose- and time-dependent manners, the IC50 value of PMB in 12, 24, 36, 48 h was 653.0 µM, 450.5 µM, 338.1 µM and 311.4 µM, respectively (Fig. 2A). Flow cytometry analysis was conducted to detect the levels of cell apoptosis. A significant time- and dose-dependently increase in apoptosis rate was observed after PMB treatment about 12 and 24 h, the percentage of early-apoptotic and late-apoptotic cells in PMB-125 µM, PMB-500 µM, PMB-1000 µM treatment about 12h groups were 3.6%±0.6%, 9.6%±2.6% and 24.3%±10.0%, respectively, and 24h were 7.7%±1.9%, 16.9%±2.5% and 36.9%±10.6%, respectively (Fig. 2B). Western blot experiment was performed to assess the expression of apoptotic pathway proteins in NRK-52E after PMB treatment. As shown in Fig. 2C-D, the expression of caspase-9 and caspase-3 proteins were increased after PMB treatment about 24 h, and the Bax/Bcl-2 ratio was also increased.
3.2 PMB induced kidney injury in mice in a dose- and time-dependent manner
To detect the toxicity of PMB in vivo, the following experiment was done. All mice were randomly divided into 4 groups with different treatments including control (0.9% saline), PMB-L (9.3mg/kg), PMB-M (14.0 mg/kg), PMB-H (18.7 mg/kg) for 3 days or 7 days. The injury markers of the liver and kidney were investigated to assess the potential toxic effect of PMB. Serum levels of BUN and Cre were estimated to assess kidney injury. Compared with no change after PMB treatment 3 days, increased serum BUN and Cre levels were found after PMB treatment 7 days. A similar tendency was also observed in the kidney function index (Fig. 3A). For liver function, PMB didn’t induce any significant effect on mice serum level of AST, ALT and TBA in 3 days or 7 days, while the serum level of ALP was increased in 7 days (p < 0.05). The liver function index was also detected and unchanged (Fig. 3B). These results indicated that PMB induced kidney injury in a dose- and time-dependent manner but had no impact on the liver. Additionally, HE stained was applied to detect injury in the liver and kidney after PMB treatment. As shown in Fig. 4, marked histopathological alterations such as tubular dilation and vacuole formation were manifested in the kidneys of mice in the group treated with 18.7 mg/kg polymyxin B (PMB-H), though only slightly tubular and glomerular injury were observed in 9.3 mg/kg PMB group (PMB-M) and 14.0 mg/kg PMB group (PMB-H).
Based on the above results, considering that PMB treatment for 3 days did not cause significant damage to kidney tissue, we mainly studied the kidney damage of mice after PMB treatment for 7 days in the follow-up experiments.
3.3 PMB promoted apoptosis in kidney tissue
Next, the apoptosis level in mice kidney tissue was explored after PMB treatment. Results from qPCR assay showed that compared with control group, the ratio of Bax/Bcl-2 mRNA expression was increased in PMB-M (p < 0.05), PMB-H (p < 0.01) in a dose-dependent manner, Caspase-9 mRNA expression was dose-dependently increased in PMB-L (p < 0.01), PMB-M (p < 0.01), PMB-H (p < 0.01), and Caspase-3 mRNA expression was dose-independently increased in PMB-M (p < 0.01) and PMB-H (p < 0.01) (Fig. 5A). Then, the expression of apoptosis-related proteins was measured by Western Blot analysis. Consistence with the results from the qPCR assay, PMB increased the ratio of Bax/Bcl-2 protein in a dose-dependent manner, the expression of Caspase-9 protein was dose-dependently increased in PMB-L (p < 0.05), PMB-M (p < 0.01), PMB-H (p < 0.01), the expression of Caspase-3 protein was increased after PMB treatment and arrived at highest in PMB-M group (p < 0.01) (Fig. 5B-C). The above results indicated that PMB promoted apoptosis in kidney tissue.
3.4 The decline of antioxidant ability and the generation of Reaction Oxidative stress was involved in PMB-induced kidney injury in vivo and in vitro
To investigate the effect of PMB on the antioxidant level in vivo and in vitro, we firstly measured the activities or levels of CAT, GSH, SOD and MDA in NRK-52E cells. NRK-52E cells were treated with PMB at three different dosages (125 µM, 500 µM, 1000 µM), the results indicated that PMB decreased activity of CAT, GSH, and SOD in a dose-dependent manner, while the content of MDA was dose-dependently increased in the PMB-125 µM group (p < 0.05), PMB-250 µM group (p < 0.01) and PMB-1000 µM group (p < 0.05) (Fig. 6A). In vivo study, after 7 days of drug exposure, decreased activity or content of CAT, GSH, and SOD were observed in PMB-L (no significant), PMB-M (p < 0.05), and PMB-H group (p < 0.05) by a dose-dependent manner, while the content of MDA was dose-independently increased in PMB treatment (p < 0.05) (Fig. 6B). Then, intracellular ROS was evaluated after PMB treatment, the results from flow cytometry analysis and fluorescence microscope showed that PMB induced the production of ROS in a dose-dependent manner (Fig. 6C-D). These results indicated that PMB declined antioxidant ability and induced the generation of ROS in vivo and in vitro, which resulted in kidney injury.
3.5 The inhibition of the Nrf2/NQO1 pathway was involved in PMB-induced toxicity
The Nrf2/NQO1 signaling pathway is an important regulator of oxidative stress. We next evaluated the Nrf2/NQO1 pathway in NRK-52E cells, western blot experiment was applied to detect the expression of Nrf2 and NQO1 protein, PMB induced dose-dependently decrease in the expression of Nrf2 and NQO1 protein (Fig. 7A). In mice study, the expression of Nrf2 and NQO1 mRNA was measured through qPCR experiments. Compared with the control group, the expression of Nrf2 mRNA was decreased after PMB-H treatment (p < 0.05), but there was no significant change in PMB-L and PMB-M groups. Besides, PMB declined the level of NQO1 mRNA in the PMB-L group (p < 0.05), PMB-M group (p < 0.01), PMB-H group (p < 0.01) with a dose-dependent manner (Fig. 7B). Western Blot experiment showed that PMB down-regulated the expressions of Nrf2 and NQO1 proteins in kidney tissues, the expression of Nrf2 protein was decreased in PMB-L (no significant), PMB-M (p < 0.05), PMB-H (p < 0.01) in a dose-dependent manner, while NQO1 protein expression was lowest in PMB-M group (p < 0.01) (Fig. 7C). These results demonstrated that the inhibition Nrf2/NQO1 pathway was involved in PMB-induced nephrotoxicity.