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Isoliquiritigenin Pretreatment Induces Endoplasmic Reticulum Stress-Mediated Hormesis and Attenuates Cisplatin-Induced Oxidative Stress and Damage in LLC-PK1 Cells

Isoliquiritigenin (IsoLQ) is a flavonoid with antioxidant properties and inducer of endoplasmic reticulum (ER) stress. In vitro and in vivo studies show that ER stress-mediated hormesis is cytoprotective; therefore, natural antioxidants and ER stress inducers have been used to prevent renal injury. Oxidative stress and ER stress are some of the mechanisms of damage involved in cisplatin (CP)-induced nephrotoxicity. This study aims to explore whether IsoLQ pretreatment induces ER stress and produces hormesis to protect against CP-induced nephrotoxicity in Lilly Laboratories Cell-Porcine Kidney 1 (LLC-PK1) cells. During the first stage of this study, both IsoLQ protective concentration and pretreatment time against CP-induced toxicity were determined by cell viability. At the second stage, the effect of IsoLQ pretreatment on cell viability, ER stress, and oxidative stress were evaluated. IsoLQ pretreatment in CP-treated cells induces expression of glucose-related proteins 78 and 94 kDa (GRP78 and GRP94, respectively), attenuates CP-induced cell death, decreases reactive oxygen species (ROS) production, and prevents the decrease in glutathione/glutathione disulfide (GSH/GSSG) ratio, free thiols levels, and glutathione reductase (GR) activity. These data suggest that IsoLQ pretreatment has a moderately protective effect on CP-induced toxicity in LLC-PK1 cells, through ER stress-mediated hormesis, as well as by the antioxidant properties of IsoLQ.


Determination of IsoLQ Protective Concentration and Pretreatment Time against CP-Induced Toxicity in LLC-PK1 Cells
According to the data shown in Figure 2, 40 μM CP caused ~66% of cell death (p < 0.05), while Figure 3 shows that 15 and 25 μM IsoLQ attenuated CP-induced decrease on cell viability (p < 0.05). Therefore, these two concentrations were used to determine the IsoLQ pretreatment time that could have a protective effect against CP-induced toxicity in LLC-PK1 cells. Cell viability results indicate that 6 and 8 h pretreatment with 15 and 25 μM IsoLQ promoted cytoprotective effects against 40 μM CP (p < 0.05) ( Figure 4).

Pretreatment of IsoLQ Induces ER Stress
According to the data shown in Figure 4, 15 and 25 μM IsoLQ pretreatment attenuated CP-induced cell death (p < 0.05), so cells were treated with the highest concentration of IsoLQ (25 μM) to evaluate ER stress. Figure 5 shows that GRP94 and GRP78 increased at 4 and 6 h (p < 0.05) respectively, after pretreatment with 25 μM IsoLQ, and remained increased at 24 h.

IsoLQ Pretreatment Enhanced ER Stress in CP-Induced Nephrotoxicity in LLC-PK1 Cells
Based on cell viability results as well as protein levels of GRP78 and GRP94 obtained from first-stage experiments, the experimental protocol was designed as follows: cells were pretreated with 25 μM IsoLQ for 8 h (the highest effects on cell viability and GRP78 and GRP94 expression were observed in these conditions) and subsequently with 40 μM CP (the highest CP concentration evaluated) for a time-course study. After pretreatment of LLC-PK1 cells with 25 μM IsoLQ for 8 h and exposure to 40 μM CP for 16 and 24 h, GRP78 and GRP94 expression increased (p < 0.05) (Figure

Pretreatment of IsoLQ Induces ER Stress
According to the data shown in Figure 4, 15 and 25 µM IsoLQ pretreatment attenuated CP-induced cell death (p < 0.05), so cells were treated with the highest concentration of IsoLQ (25 µM) to evaluate ER stress. Figure 5 shows that GRP94 and GRP78 increased at 4 and 6 h (p < 0.05) respectively, after pretreatment with 25 µM IsoLQ, and remained increased at 24 h.

Pretreatment of IsoLQ Induces ER Stress
According to the data shown in Figure 4, 15 and 25 μM IsoLQ pretreatment attenuated CP-induced cell death (p < 0.05), so cells were treated with the highest concentration of IsoLQ (25 μM) to evaluate ER stress. Figure 5 shows that GRP94 and GRP78 increased at 4 and 6 h (p < 0.05) respectively, after pretreatment with 25 μM IsoLQ, and remained increased at 24 h.

IsoLQ Pretreatment Enhanced ER Stress in CP-Induced Nephrotoxicity in LLC-PK1 Cells
Based on cell viability results as well as protein levels of GRP78 and GRP94 obtained from first-stage experiments, the experimental protocol was designed as follows: cells were pretreated with 25 μM IsoLQ for 8 h (the highest effects on cell viability and GRP78 and GRP94 expression were observed in these conditions) and subsequently with 40 μM CP (the highest CP concentration evaluated) for a time-course study. After pretreatment of LLC-PK1 cells with 25 μM IsoLQ for 8 h and exposure to 40 μM CP for 16 and 24 h, GRP78 and GRP94 expression increased (p < 0.05) (Figure

IsoLQ Pretreatment Enhanced ER Stress in CP-Induced Nephrotoxicity in LLC-PK1 Cells
Based on cell viability results as well as protein levels of GRP78 and GRP94 obtained from first-stage experiments, the experimental protocol was designed as follows: cells were pretreated with 25 µM IsoLQ for 8 h (the highest effects on cell viability and GRP78 and GRP94 expression were observed in these conditions) and subsequently with 40 µM CP (the highest CP concentration evaluated) for a time-course study. After pretreatment of LLC-PK1 cells with 25 µM IsoLQ for 8 h and exposure to 40 µM CP for 16 and 24 h, GRP78 and GRP94 expression increased (p < 0.05) ( Figure 6A). This increase was higher than that under IsoLQ treatment or with CP alone (Figures 5 and 6B, respectively). In addition, the expression of the eukaryotic initiation factor 2 alpha (p-eIF2α) did not change (data not shown).
Molecules 2020, 25, x FOR PEER REVIEW 5 of 16 6A). This increase was higher than that under IsoLQ treatment or with CP alone (Figures 5 and 6B, respectively). In addition, the expression of the eukaryotic initiation factor 2 alpha (p-eIF2α) did not change (data not shown).

IsoLQ-Induced Hormesis Attenuates Oxidative Stress in CP-Treated LLC-PK1 Cells
LLC-PK1 cells were pretreated with 25 μM IsoLQ for 8 h and then with 40 μM CP for 16 and 24 h, as described for the second stage of the experimental design. 25 μM IsoLQ pretreatment decreased ROS production by ~37% and ~26% after exposure to 40 μM CP for 16 and 24 h (p < 0.05) respectively, and attenuated the decrease in GSH/glutathione disulfide (GSSG) ratio, free thiols, and glutathione reductase (GR) activity 24 h after CP treatment (p < 0.05) ( Table 1).

IsoLQ-Induced Hormesis Attenuates Oxidative Stress in CP-Treated LLC-PK1 Cells
LLC-PK1 cells were pretreated with 25 µM IsoLQ for 8 h and then with 40 µM CP for 16 and 24 h, as described for the second stage of the experimental design. 25 µM IsoLQ pretreatment decreased ROS production by~37% and~26% after exposure to 40 µM CP for 16 and 24 h (p < 0.05) respectively, and attenuated the decrease in GSH/glutathione disulfide (GSSG) ratio, free thiols, and glutathione reductase (GR) activity 24 h after CP treatment (p < 0.05) ( Table 1). In order to determine intracellular ROS sources, we used diphenyleneiodonium chloride (DPI), an inhibitor of nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase (NOX), or N-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS. DPI did not decrease ROS production in any of the groups of treated cells, whereas L-NAME decreased ROS production at 16 h in IsoLQ + CP-treated cells to similar levels as the control group (p < 0.05). This effect remained until 24 h (p < 0.05) ( Figure 7); whereas in CP-treated cells, ROS production decreased only at 16 h (p < 0.05).
Molecules 2020, 25, x FOR PEER REVIEW 6 of 16 In order to determine intracellular ROS sources, we used diphenyleneiodonium chloride (DPI), an inhibitor of nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase (NOX), or N-nitro-L-arginine methyl ester (L-NAME), an inhibitor of NOS. DPI did not decrease ROS production in any of the groups of treated cells, whereas L-NAME decreased ROS production at 16 h in IsoLQ + CP-treated cells to similar levels as the control group (p < 0.05). This effect remained until 24 h (p < 0.05) ( Figure 7); whereas in CP-treated cells, ROS production decreased only at 16 h (p < 0.05).

Discussion
IsoLQ is a flavonoid with anti-inflammatory, antitumoral, and antioxidant properties [1][2][3][4][5][6][7]16,59] that exerts protective effects in liver and kidney cells [3,4,9,[16][17][18]. In addition, it has been demonstrated that IsoLQ does not interfere with CP antineoplastic activity [16]. The effect of IsoLQ on cell viability in CP-induced nephrotoxicity was a biphasic dose-response: low IsoLQ concentrations attenuated the decrease on cell viability, but high IsoLQ concentrations inhibited it. This effect is known as hormesis, a term that refers to a biological favorable cellular response [40,[47][48][49][50]60]. The IsoLQ + CP dose-response curve showed an inverted J-shaped hormetic effect and this biphasic curve has an interval called the hormetic zone where the beneficial effects are evident ( Figure 3) [45,[47][48][49]. The low-dose stimulation reflects the capacity of the cells to distribute biological resources that help to defend the organism from a wide range of stressor agents, such as CP, and this adaptive mechanism includes stress protein responses that reduce the damage more effectively than the response induced only by exposure to the stressor [40,41,46,47]. Therefore, hormesis is more than simply a dose-response relationship, it is a quantitative and temporal manifestation of reparative processes that is adaptive in nature, such as UPR elicited by ER stress [42,43,46,47,50,60,61].
Data obtained show that 25 µM IsoLQ pretreatment induced ER stress and also attenuated 40 µM CP-induced cell death in LLC-PK1 cells. The increase in the expression of the chaperones GRP78 and GRP94 indicates the occurrence of ER stress and suggests the activation of UPR [27,29,37,[62][63][64][65]. The early induction of ER stress could play a key role in cell survival, because ER stress induced by IsoLQ pretreatment generated a hormetic effect to activate adaptive responses such as UPR, that protects against higher stress levels as occurs in CP-induced nephrotoxicity. In in vitro and in vivo models of kidney diseases, like renal ischemia-reperfusion or exposure to cytotoxic compounds, the a priori induction of ER stress with pharmacological or natural compounds generates tolerance against these insults (hypoxia, oxidative stress) in order to inhibit the apoptotic cell death [37,52,53,62,[66][67][68][69].
On the other hand, oxidative stress is also characteristic in CP-induced nephrotoxicity and is an important factor in the development and progression of other associated damage mechanisms, such as mitochondrial dysfunction, inflammation, and ER stress [19][20][21][22]25,26,54,70]. ROS are generated naturally and regulate cell functions [71,72]. However, excessive quantities of ROS can result in damage to proteins, modifying their function and disrupting ER homeostasis [27,32,34,73]. ROS overproduction can be sensed by the ER, which is highly sensitive to changes in ROS levels through redox sensors, such as, for example, the thiol groups of cysteines [33,70,[74][75][76][77], causing misfolded protein accumulation and consequently increasing ROS production, which leads to a vicious cycle able to activate UPR, generate chronic stress, and induce apoptosis [32][33][34]70,72]. Moreover, ROS overproduction causes a decrease in the GSH/GSSG ratio and in antioxidant enzyme levels, whilst oxidizing lipids, DNA, and proteins [19][20][21][22]25,26]. In our experimental model, ROS production could be associated to NOS activity and consequently to overproduction of O 2 •− that enhances oxidative stress. However, IsoLQ can attenuate this oxidative imbalance due to its ROS scavenging capacity [4,7]. In CP-induced nephrotoxicity, the decrease in the GSH/GSSG ratio is characteristic due to the formation of GSH-CP adducts. The positively charged species of CP (their active forms) have a great affinity for thiol groups [20,22,25,78,79] and consequently, there is an alteration in the activity of enzymes related to GSH metabolism [19,26,78]. In addition, the decrease in GSH could be associated with the formation of disulfide bonds among misfolded proteins in a coupled reaction with the protein disulfide isomerase and ER oxidoreductase 1 [73,[80][81][82][83]. Interestingly, ER stress induction is also associated with an increase in GSH biosynthesis in order to form disulfide bonds between proteins and recycle unfolded proteins. This effect could explain the increase in free thiols, such as GSH, in cells pretreated with IsoLQ [33,82]. Moreover, it has been described that ER stress increases the activity of antioxidant enzymes such as GR, glutathione peroxidase, and glutathione S-transferase [33,63,82]. Probably, augmented GR activity in cells pretreated with IsoLQ was due to this effect, since IsoLQ is also a bifunctional antioxidant able to induce expression of phase II detoxifying enzymes through nuclear factor erythroid 2 (Nrf2) transcriptional regulation [9,10]. Under ER stress, protein kinase ribonucleic acid (RNA)-activated-like ER kinase (PERK), one the of the signaling pathways of UPR, directly phosphorylates Nrf2, causing Nrf2 to dissociates from its negative regulator (Kelch-like erythroid-derived cap-n-collar homology-(ECH-)associated protein 1); after which, Nrf2 translocates to the nucleus, leading the transcription of genes that encode phase II detoxifying enzymes that maintain redox homeostasis and may contribute to cell survival [84][85][86]. In addition, eIF2α is another target of the PERK signaling pathway [27,29,63,65]. Even though the data on p-eIF2α expression are inconclusive (data not shown), it is possible that the PERK pathway has become activated. Nevertheless, studies on the role of UPR proteins are required to describe and understand the mechanisms involved in the hormetic effect produced by IsoLQ in this experimental model. In summary, Figure 8 shows a possible mechanism of the protective effect of IsoLQ against CP-induced nephrotoxicity in LLC-PK1 cells. We derived our assumptions from the main observations found in this study. maintain redox homeostasis and may contribute to cell survival [84][85][86]. In addition, eIF2α is another target of the PERK signaling pathway [27,29,63,65]. Even though the data on p-eIF2α expression are inconclusive (data not shown), it is possible that the PERK pathway has become activated. Nevertheless, studies on the role of UPR proteins are required to describe and understand the mechanisms involved in the hormetic effect produced by IsoLQ in this experimental model. In summary, Figure 8 shows a possible mechanism of the protective effect of IsoLQ against CP-induced nephrotoxicity in LLC-PK1 cells. We derived our assumptions from the main observations found in this study.

First Stage
The aim of the first stage set of experiments was to determine the potential IsoLQ protective concentration and pretreatment time against CP-induced toxicity in LLC-PK1 cell culture. Cells were treated with CP (10-80 µM) for 24 h to calculate the IC 50 . Additionally, cells were pretreated with IsoLQ (5-80 µM) for 6 h and later incubated with one concentration higher than the IC 50 of CP (35 µM) for 24 h to evaluate the potentially hormetic dose-response curve for cell viability. Finally, two IsoLQ concentrations within the hormetic zone (15 and 25 µM) and one concentration higher than the IC 50 of CP (40 µM) were selected to test the putative protective effect by IsoLQ on cell viability.
On the other hand, cells were pretreated with 25 µM IsoLQ (highest concentration within the hormetic zone) in a time-course study (2-24 h) to evaluate the expression of GRP78 and GRP94.

Second Stage
Based on the first stage results, LLC-PK1 cells were pretreated with 25 µM IsoLQ for 8 h and subsequently incubated, in a time-course study, with 40 µM CP to evaluate ER stress (2, 16, and 24 h) and oxidative stress (16 and 24 h). The experimental design is summarized in Figure 9.

Cell Viability
Subsequently after treatments, DMEM was removed and cells were washed with phosphatebuffered saline (PBS) followed by the addition of 12 µM FDA and incubation for 5 min at 37 • C in darkness [88]. Fluorescence was determined by a microplate reader at excitation and emission wavelengths of 485 and 520 nm, respectively. The results are expressed as the percentage of the control (without treatments).

Cell Viability
Subsequently after treatments, DMEM was removed and cells were washed with phosphate-buffered saline (PBS) followed by the addition of 12 μM FDA and incubation for 5 min at 37 °C in darkness [88]. Fluorescence was determined by a microplate reader at excitation and emission wavelengths of 485 and 520 nm, respectively. The results are expressed as the percentage of the control (without treatments).

ER Stress Evaluation
Western Blot Analysis Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride membranes, which were blocked for one hour with 5% skimmed milk or bovine serum albumin. Membranes were left incubating overnight at 4 °C with the appropriate primary antibodies against GRP78 (1:1000), GRP94 (1:1000), and α-tubulin (1:8000). Subsequently, membranes were washed and incubated with the appropriate secondary antibodies for two hours.

Western Blot Analysis
Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to polyvinylidene fluoride membranes, which were blocked for one hour with 5% skimmed milk or bovine serum albumin. Membranes were left incubating overnight at 4 • C with the appropriate primary antibodies against GRP78 (1:1000), GRP94 (1:1000), and α-tubulin (1:8000). Subsequently, membranes were washed and incubated with the appropriate secondary antibodies for two hours. The proteins of interest were detected with the LI-COR Odyssey Sa Infrared Imaging System (Lincoln, NE, USA). Image analysis was performed with Image Studio Lite Software version 5.2.

Measurement of ROS Production
After the second stage cell treatments, DMEM was removed and cells were washed with PBS followed by incubation with 10 µM H 2 DCFDA for 30 min at 37 • C in the darkness. Fluorescence intensity was visualized and determined in a Cytation 5 Cell Imaging Reader (Biotek Instruments, Inc., Winooski, VT, USA) at excitation and emission wavelengths of 485 and 520 nm, respectively. Relative fluorescence intensity of treated cells was expressed as a percentage of the control (without treatments).
In order to determine the intracellular ROS sources, LLC-PK1 cells were pretreated with IsoLQ for 8 h, then with CP for 16 or 24 h. At 30 min before the end of the CP treatments, the NOX inhibitor 10 µM DPI and the NOS inhibitor 1 mM L-NAME, were added separately to the cell cultures. Finally, the growth medium was removed, and the measurement of ROS was carried out as described above.

Measurement of GSH/GSSG Ratio
The redox status, expressed as the GSH/GSSG ratio, was determined following a method previously described [90].

Free Thiols Levels
Free thiols were determined following a method previously described [91]. Total protein content was determined by the Bradford assay [89]. Results are expressed as nmol/mg protein.

GR Activity
Activity of GR was determined by measuring the disappearance of NADPH at 340 nm. Total protein content was determined by the Bradford assay [89]. Results are expressed as U/mg protein, where U equals one µmol of oxidized NADPH per minute [92].

Statistical Analysis
The results are expressed as mean ± standard error of the mean (SEM). Statistical analysis was performed for each determination using the software Graph-Pad Prism 6.0 (San Diego, CA, USA) comparing the mean values through a one-way analysis of variance, followed by the Tukey's multiple comparison test. For the time-course studies of Western blot, a Student's t-test was carried out with respect to the control. A p-value < 0.05 was considered significant.

Conclusions
The mechanism of cisplatin toxicity is complex and involves many cellular targets, some of which are less studied, such as ER stress, which could have cytoprotective or cytotoxic effects. However, if ER stress is induced early with natural compounds such as IsoLQ, the effects could be beneficial because the cell adapts to stress, distributing biological resources to help defend the organism from a wide range of stressor agents, such as CP. However, to describe the mechanisms involved in this specific hormetic effect, it is necessary to study and explore each UPR pathway activated in this particular experimental model.
In conclusion, our results suggest that pretreatment with IsoLQ induces the expression of GRP78 and GRP94, consequently inducing ER stress-mediated hormesis and ameliorating oxidative stress, both of which have a moderately protective effect against CP-induced nephrotoxicity in LLC-PK1 cells. This study provides another strategy or perspective to prevent cisplatin-induced toxicity, which can be applied to the study of other reticulum stress-inducing compounds.