Introduction

Drug-induced nephrotoxicity often occurs in long-term drug therapy1. The nephrotoxicity is difficult to be predicted in the early stage of disease2. At the stage of drug development, the incidence of acute kidney injury caused by drugs in the intensive care unit is close to 30–50%3. Gentamicin (GM) is an aminoglycoside drug, used to treat bacterial infections for those caused by Gram-negative organisms4. Despite its vast therapeutic benefits as an antibiotic drug, its clinical use is limited majorly because of its nephrotoxicity effect5). After screening different components of the protein, the strip identification and KEGG analysis were carried out. It was found that the structure of E7D7Z1 protein was related to cellular component, molecular function and TGF-beta signaling pathway, and it was found that both E7D7Z1 protein was contained in both SDAP1 and SDAP2. Therefore, proteomic analysis was performed on E7D7Z1 (Table 4). We selected sheep's decoration protein (W5Q2F5) as the control protein, and selected the deer decoration protein (E7D7Z1) as the model arrangement (Fig. 5A) and the basic layout (Fig. 5B). Phylogenetic analysis showed that E7D7Z1 was close to P28654 in Mus musculus (Fig. 5C). Use SWISS-MODEL's Decorin template to simulate 3D modeling and view it with Discovery Studio 4.5 (https://swissmodel.expasy.org/interactive).

Figure 1
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Isolation of SDAPR: elution profile of SDAPR by Sephadex G-100 column.

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Figure 2
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Native-PAGE of different components of SDAPR, SDAP1 and SDAP2.

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Figure 3
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LC–MS/MS images of SDAP1 and SDAP2. (A) SDAP1. (B) SDAP2.

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Table 1 Protein identification results of SDAP1.
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Table 2 Protein identification results of SDAP2.
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Figure 4
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GO and KEGG analysis of SDAPR. (A) GO analysis of SDAPR: The horizontal axis provides description of the matched GO terms, whereas the vertical axis is the ratio of proteins in the total identified proteins. (B) KEGG analysis of SDAPR: KEGG pathway annotation of tissue-enriched proteins. The horizontal axis is the number of proteins, whereas the vertical ordinates are the terms of the KEGG pathways. KEGG reference source is: https://www.kegg.jp/kegg/kegg1.html.

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Table 3 GO analysis results of SDAP1 and SDAP2.
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Figure 5
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Protein Sequence alignment, 3D modeling and phylogenetic analysis of Decorin from SDAPR. (A) A presumed sequence W5Q2F5 aligned with the model Decorin. At the bottom of columns, asterisks (*) show conserved positions, colons (:) show conserved substitutions and points (.) show non-conserved substitutions. Grey line, green bend, blue banded arrowhead and red solenoid represent coil, turn, sheet and helix, respectively. Different fragments are framed by red lines. (B) 3D modeling was simulated using the template Decorin by SWISS-MODEL and viewed by Discovery Studio 4.5. The colors grey, green, blue and red represent coils, turns, sheets and helices, respectively. Different fragments are indicated by red arrows. (C) Phylogenetic tree constructed using putative Decorin and 13 other sequences from different species using MEGA 7 with the Neighbor-Joining method.

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Table 4 KEGG analysis results of SDAP1 and SDAP2.
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Renal function and pathology damages

To investigate whether SDAP1 and SDAP2 can improve GM-induced nephrotoxicity in vivo, we established a mouse model of acute gentamicin nephrotoxicity. As shown in Table 5, compared with the control group, GM significantly reduced the weight of mice and significantly increased the kidney index. In contrast to the GM model group, SDAP1 and SDAP2 pre-protection groups partially increased body weight and reduced GM-induced increase in renal index (P < 0.01).

Table 5 SDAP1 and SDAP2 against GM-induced kidney dysfunction.
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As shown in Fig. 6A and B, compared with control mice, the levels of renal function indicators (Cr and BUN) in the serum of model mice were significantly increased (P < 0.01). Compared with model mice, it was found that SDAP1 and SDAP2 at low and high doses can significantly reduce Cr content, and SDAP1 and SDAP2 at high doses have significant effects. After the detection of BUN content, it was found that low and high doses of SDAP1 and SDAP2 can significantly reduce the content of BUN, and the effects of high doses of SDAP1 and SDAP2 were significant (P < 0.01).

Figure 6
figure 6

Effects of SDAP1 and SDAP2 treatment on GM-induced serum nephrotoxicity biomarkers in mice. Mice were pretreated with SDAP1 (15, 60 mg/kg) and SDAP2 (15, 60 mg/kg) with or without GM (100 mg/kg) for 24 h. (A) BUN levels in serum. (B) Cr levels in serum. Data are presented as the Mean ± SD (n = 5). **P < 0.01 versus control group. #P < 0.05, ##P < 0.01 versus GM group.

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The HE staining of the kidneys revealed that the kidney tissue of mice in the control group had a more normal appearance. In the model group, there was obvious edema in the interstitial space of the kidney, severe glomerular shrinkage, and obstruction of the renal tubular cavity, and some cells were swollen and vacuolated. In the SDAP1 and SDAP2 low- and high-dose groups, the renal tubular tissue showed reduced focal lesions, renal tubular swelling and vacuole degeneration were also improved, and inflammatory cell infiltration and interstitial edema were alleviated to varying degrees. SDAP1 and SDAP2 improved better at high doses. The results are shown in Fig. 7.

Figure 7
figure 7

Effect of SDAP1 and SDAP2 treatment on GM-induced histopathology change in mice. (a) Control; (b) GM (100 mg/kg); (c) SDAP1 (15 mg/kg) + GM (100 mg/kg); (d) SDAP1 (60 mg/kg) + GM (100 mg/kg); (e) SDAP2 (15 mg/kg) + GM (100 mg/kg); (f) SDAP2 (60 mg/kg) + GM (100 mg/kg); (g) mouse kidney tissue semi-quantitative score. Images are shown at a magnification of 200 × . Data are mean ± SD (n = 3). **P < 0.01 as compared to the Control group. ##P < 0.01 as compared to the GM-treated group.

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Semi quantitative analysis of renal tubulointerstitial injury score: inflammatory cell infiltration < 25% is 1 score, 25–49% is 2 score, 50–75% is 3 score, and > 75% is 4 score; renal interstitial edema is 1 score of mild, 2 score of severe; renal tubulointerstitial injury is only 1 score of epithelial cell vacuole degeneration, 2 score of brush border, 3 score of necrosis. The semi quantitative renal tubulointerstitial score is the sum of the above scores (Fig. 7g).

Oxidative stress and antioxidant defense

In HEK293 cells, GM decreased the level of antioxidants such as GSH (Fig. 8A), CAT (Fig. 8B) and SOD (Fig. 8C), while increased the production of MDA (Fig. 8D) and LDH (Fig. 8E). Conversely, SDAPR (1, 2, 4 mg/mL), SDAP1 and SDAP2 (0.25, 0.5, 1 mg/mL) reversed the decrease in HEK293 cells SOD, GSH and GSH, and the overproduction of MDA and LDH. Excessive ROS induced by harmful stimulation will cause oxidative damage to biomolecules (such as lipids, DNA, proteins, etc.), thus inducing cell injury or death. The changes in intracellular ROS were detected by DCFH-DA staining. Damaged cells exerted strong fluorescent intensity, which indicated that massive ROS generated. HEK-293 cells were treated with GM and SDAPR, SDAP1, SDAP2 for 24 h and ROS level was detected using the ROS fluorescent probe DCFH-DA. As shown in Fig. 9, SDAPR, SDAP1 and SDAP2 treatment obviously decreased GM-induced intracellular ROS accumulation.

Figure 8
figure 8

Effect of SDAPR, SDAP1 and SDAP2 treatment on GM-induced the oxidative stress parameters in HEK 293. (A) GSH levels in HEK293. (B) CAT activity in HEK293. (C) SOD activity in HEK293. (D) MDA levels in HEK293. (E) LDH content in HEK293. Data are means ± SD (n = 4). **P < 0.01 as compared to the Control group. ##P < 0.01 as compared to the GM-treated group.

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Figure 9
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SDAPR, SDAP1 and SDAP2 significantly suppresses ROS production of HEK‑293 cells induced with GM. (A) Control; (B) GM (3 mg/mL); (C) SDAPR (1 mg/mL) + GM (3 mg/mL); (D) SDAPR (2 mg/mL) + GM (3 mg/mL); (E) SDAPR (4 mg/mL) + GM (3 mg/mL); (F) SDAPR (0.25 mg/mL) + GM (3 mg/mL); (G) SDAPR (0.5 mg/mL) + GM (3 mg/mL); (H) SDAPR (1 mg/mL) + GM (3 mg/mL); (I) SDAPR (0.25 mg/mL) + GM (3 mg/mL); (J) SDAPR (0.5 mg/mL) + GM (3 mg/mL); (K) SDAPR (1 mg/mL) + GM (3 mg/mL); (L) Relative expression of ROS in HEK293 cells. Data are means ± SD (n = 3). **P < 0.01 as compared to the Control group. ##P < 0.01 as compared to the GM-treated group.

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Mitochondrial dysfunction and apoptosis

In order to maintain mitochondrial function, balancing mitochondrial membrane potential is critical. Compare with the control group, HEK293 cells of GM group reduced the production red fluorescence meanwhile increased the intensity of green fluorescence (P < 0.01). By contrast, SDAPR (1.0, 2.0 and 4.0 mg/mL) significantly increased the red/green fluorescence ratio of HEK293 cells which pretreated with SDAPR (P < 0.01 and P < 0.01, respectively). Our data suggested that SDAPR pretreatment reverses mitochondrial depolarization and maintains mitochondrial function (Fig. 10).

Figure 10
figure 10

Effects of SDAPR on GM induced mitochondrial membrane potential (ΔΨm) depolarization in HEK-293 cells (scalebar, 50 μm). (A) Control; (B) GM (3 mg/mL); (C) SDAPR (1 mg/mL) + GM (3 mg/mL); (D) SDAPR (2 mg/mL) + GM (3 mg/mL); (E) SDAPR (4 mg/mL) + GM (3 mg/mL); (F) Red/green fluorescence intensity of JC-10 in HEK293 cells. Data are presented as the Mean ± SD (n = 6). **P < 0.01 versus control group. ##P < 0.01 versus GM group. SDAPR Sika deer antler protein, GM gentamicin, HEK293 human embryonic kidney 293.

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MTT and RTCA experiments were performed to study the effects of SDAPR, SDAP1 and SDAP2 on the viability of GM-induced HEK293 cells. To screen the pre-protection of SDAP1 and SDAP2, cells were pretreated with SDAP1 and SDAP2 (0.25, 0.5, 0.75, 1, 1.25, 1.5 mg/mL) before administration of GM (3 mg/mL) for 24 h (Fig. 11A). We selected SDAP1 and SDAP2 at 0.25, 0.5, and 1 mg/mL for subsequent experiments. Compared with the control group, GM (3 mg/mL) significantly reduced cell viability. In addition, the other groups pretreated with SDAPR (1, 2, 4 mg/mL), SDAP1 (0.25, 0.5, 1 mg/mL), and SDAP2 (0.25, 0.5, 1 mg/mL) for 24 h had significant differences compared with the GM group (P < 0.01). These results indicate that SDAPR, SDAP1 and SDAP2 provide dose-dependent protection against GM-induced cell damage (Fig. 11B). As shown in Fig. 11C, RTCA experiments confirmed that SDAP1 (1 mg/mL) and SDAP2 (1 mg/mL) were more protective than SDAPR (4 mg/mL).

Figure 11
figure 11

Effect of different components of SDAPR on GM-induced cytotoxicity in HEK-293 cells. (A) Cells were pretreated with SDAP1 and SDAP2 (0.25, 0.5, 0.75, 1, 1.25, 1.5 mg/mL) before administration of GM (3 mg/mL) for 24 h. Data are means ± SD (n = 6). (B) Cells were pretreated with SDAPR (1, 2, 4 mg/mL), SDAP1 and SDAP2 (0.25, 0.5, 1 mg/mL) before administration of GM (3 mg/mL) for 24 h. Data are means ± SD (n = 6). **P < 0.01 as compared to the Control group. ##P < 0.01 as compared to the GM-treated group. (C) Cells were pretreated with SDAPR (4 mg/mL), SDAP1 and SDAP2 (1 mg/mL) before administration of GM (3 mg/mL) for 24 h. Data are means ± SD (n = 3). **P < 0.01 as compared to the Control group. ##P < 0.01 as compared to the GM-treated group.

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In order to investigate whether GM-mediated toxicity of HEK-293 cells is related to the activation of apoptotic cells, flow cytometry was used to detect apoptotic cells. As shown in Fig. 12, compared with the control group, the total apoptotic cells (early and late apoptotic) increased significantly to 51.29% (P < 0.01) in the GM treatment group of 3 mg/mL for 24 h. However, after pretreatment with SDAPR (1 mg/mL, 2 mg/mL and 4 mg/mL), the apoptotic cells decreased from 51.29 to 38.32%, 26.43% and 24.80% respectively. Moreover, after SDAP1 (0.25 mg/mL, 0.5 mg/mL and 1 mg/mL) pretreatment, the apoptotic cells decreased from 51.29 to 34.40%, 24.66% and 12.82% respectively. At the same time, SDAP2 (0.25 mg/mL, 0.5 mg/mL and 1 mg/mL) was used to protect the apoptotic cells from 51.29 to 35.11%, 26.17% and 23.62% respectively. The results showed that although GM treatment resulted in increased apoptosis, the combination of SDAPR, SDAP1 and SDAP2 had the opposite effect on GM-induced apoptosis (Fig. 12A). In addition, we also used Hoechst 33258 staining to determine the effects of SDAPR, SDAP1 and SDAP2 on GM-induced apoptosis of HEK-293 cells. In Fig. 13B, the results showed that compared with the control group, Hoechst 33258 staining positive cells of HEK293 cells in GM group increased, and the morphology of typical apoptotic cells such as chromatin agglutination (bright blue fluorescence), fragmentation and marginalization increased. Compared with GM group, the Hoechst33258 staining positive cells of HEK293 cells in the SDAPR, SDAP1 and SDAP2 administration groups decreased, and the typical apoptotic cell morphology such as chromatin condensation (bright blue fluorescence), fragmentation and marginalization was alleviated with increasing drug concentration (Fig. 12B).

Figure 12
figure 12

Effect of SDAPR, SDAP1 and SDAP2 on apoptosis induction in GM treated HEK-293 cells. (A) Result of the apoptosis in HEK293 cells were analyzed by flow cytometry. (B) The nuclear morphology of HEK293 cells were evaluated using Hoechst 33,258 staining. (a) Control; (b) GM (3 mg/mL); (c) SDAPR (1 mg/mL) + GM (3 mg/mL); (d) SDAPR (2 mg/mL) + GM (3 mg/mL); (e) SDAPR (4 mg/mL) + GM (3 mg/mL); (f) SDAPR (0.25 mg/mL) + GM (3 mg/mL); (g) SDAPR (0.5 mg/mL) + GM (3 mg/mL); (h) SDAPR (1 mg/mL) + GM (3 mg/mL); (i) SDAPR (0.25 mg/mL) + GM (3 mg/mL); (j) SDAPR (0.5 mg/mL) + GM (3 mg/mL); (k) SDAPR (1 mg/mL) + GM (3 mg/mL). Data are presented as the Mean ± SD (n = 3). **P < 0.01 versus control group. #P < 0.05, ##P < 0.01 versus GM group.

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Figure 13
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Detection of Bax, Bcl-2, Cleaved-Caspase 3 and P53 protein expression by Western blotting. (A) Immunoblotting for the target molecule of Bax, Bcl-2, Cleaved-Caspase 3 and Caspase 3 showing the effects of SDAPR, SDAP1 and SDAP2 in GM treated HEK-293 cells; (B) expression of Bax was determined by western blotting; (C) expression of Bcl-2 was determined by western blotting; (D) expression of Cleaved-caspase 3 was determined by western blotting; (E) expression of P53 was determined by western blotting. Data are means ± SD (n = 3). **P < 0.01 as compared to the Control group. ##P < 0.01 as compared to the GM-treated group.

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At the same time, we used Western blot analysis to detect the expression of apoptosis-related proteins in vivo and in vitro. The results showed that treatment of HEK293 cells with GM reduced the expression of the anti-apoptotic protein BCL-2, and the administration of SDAPR, SDAP1 and SDAP2 significantly decreased the expression of BCL-2. We also evaluated the expression of pro-apoptotic Bax and found that its expression was significantly increased after GM treatment (P < 0.01), however, with the addition of SDAPR, SDAP1 and SDAPR significantly reduced Bax expression. At the same time, the expression of cleaved-caspase-3 was also evaluated. Western blot analysis showed that SDAPR, SDAP1 and SDAP2 could reverse the increase of GM-induced cleaved-caspase-3 protein expression in HEK293 cells. In addition, p53 expression was also increased after GM treatment compared with the control group (P < 0.01). SDAPR, SDAP1 and SDAP2 attenuate GM-induced increase in p53 expression (Fig. 13). In vivo, we evaluated the expression of cleaved-Caspase-3 using Western blot analysis (Fig. 14). Western blot analysis showed that SDAP1 and SDAP2 can reverse the increase in Caspase-3 cleavage protein expression. In addition, compared with the control group, p53 expression was also increased after GM treatment (P < 0.01). SDAP1 and SDAP2 attenuated the GM-induced increase in p53 expression.

Figure 14
figure 14

Western blot was used to detect the expression of Cleaved-Caspase 3 and P53 protein. (A) Western blot of Cleaved-Caspase 3 and P53 target molecules, showing the role of SDAP1 and SDAP2 in kidneys of GM-treated mice; (B) Western blot to determine the expression of Cleaved-caspase 3; (C) Western blot to determine the expression of P53 expression. Data are mean ± SD (n = 3). **P < 0.01 as compared to the Control group. ##P < 0.01 as compared to the GM-treated group.

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Nrf2, NF-κB and PI3K analysis

To investigate the molecular basis of a protective effect of SDAPR against GM-induced HEK293 cells damage. Figure 15A is a Western blot of nuclear Nrf2, total HO-1, total keap1 and total NQO1. We examined whether SDAPR, SDAP1 and SDAP2 treatment affected HEK293 cells expression of nuclear Nrf2 (Fig. 15B), total HO-1 (Fig. 15C), total keap1 (Fig. 15D) and total NQO1 (Fig. 15E). GM markedly reduced expression of nuclear Nrf2, total HO-1, and total NQO1 (P < 0.01). However, compared with the group receiving only GM, SDAPR, SDAP1 and SDAP2 significantly reversed these effects (P < 0.01). In addition, the expression of total Keap1 protein was significantly increased in GM group, which was reversed by SDAPR, SDAP1 and SDAP2 (P < 0.01). Figure 16A is a Western blot of nuclear Nrf2, total HO-1 and total NQO1 in vivo. At the same time, it was investigated whether SDAP1 and SDAP2 treatment affected the expression of Nrf2 (Fig. 16B), total HO-1 (Fig. 16C), and total NQO1 (Fig. 16D) in the renal nucleus of mice. GM significantly reduced the expression of nuclear Nrf2, total HO-1 and total NQO1 (P < 0.01). However, SDAP1 and SDAP2 significantly reversed these effects compared to the group receiving only GM (P < 0.01).

Figure 15
figure 15

Detection of Nrf2, HO-1, keap1, NQO1 protein expression by Western blotting. (A) Immunoblotting for the target molecule of Nrf2, HO-1, keap1 and NQO1 showing the effects of SDAPR, SDAP1 and SDAP2 in GM treated HEK-293 cells; (B) expression of Nrf2 was determined by western blotting; (C) expression of HO-1 was determined by western blotting; (D) expression of keap1 was determined by western blotting; (E) expression of NQO1 was determined by western blotting. Data are means ± SD (n = 3). **P < 0.01 as compared to the Control group. ##P < 0.01 as compared to the GM-treated group.

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Figure 16
figure 16

Western blot was used to detect the expression of Nrf2 pathway protein. (A) Western blot of Nrf2, HO-1 and NQO1; (B) Western blot to determine the expression of Nrf2; (C) Western blot to determine the expression of HO-1; (D) Western blot to determine the expression of NQO1. Data represent mean ± SD (n = 3). **P < 0.01 as compared to the Control group. ##P < 0.01 as compared to the GM-treated group.

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Here, we validated the effect of SDAPR, SDAP1 and SDAP2 on TNF-α, IL-6 and NF-κB in GM-induced inflammation. The results showed that the production of inflammatory cytokines TNF-α and IL-6 increased in the GM group, while the production of TNF-α and IL-6 in the SDAPR, SDAP1 and SDAP2 treatment groups decreased (P < 0.01). In addition, SDAPR, SDAP1 and SDAP2 also significantly reduced GM-induced NF-κB expression in HEK293 cells (Fig. 17).

Figure 17
figure 17

Determination of inflammation index in HEK293 cells. (A) Immunoblotting for the target molecule of NF-κB showing the effects of SDAPR, SDAP1 and SDAP2 in GM treated HEK-293 cells; (B) expression of NF-κB was determined by western blotting; (C) Elisa assay for TNF-α in HEK293 cells; (D) Elisa assay for IL-6 in HEK293 cells. Data are means ± SD (n = 3). **P < 0.01 as compared to the Control group. ##P < 0.01 as compared to the GM-treated group.

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We hypothesized that SDAPR, SDAP1 and SDAP2 against GM-induced oxidative stress and apoptosis effects may be related to PI3K/AKT Pathway by KEGG analysis (Fig. 4B). Therefore, we examined the expression of PI3K, p-PI3K, Akt, p-Akt, in each group of HEK293 cells by Western blot (Fig. 18). Compared with the control group, the expression of p-PI3K and p-Akt were significantly lower in the GM model group (P < 0.01). SDAPR (1, 2, 4 mg/mL), SDAP1 (0.25, 0.5, 1 mg/mL) and SDAP2 (0.25, 0.5, 1 mg/mL) pretreatment dose-dependently increased the phosphorylation of PI3K and Akt (P < 0.01).

Figure 18
figure 18

Detection of PI3K, p-PI3K, AKT, p-AKT protein expression by Western blotting. (A) Immunoblotting for the target molecule of PI3K, p-PI3K, AKT, and p-AKT showing the effects of SDAPR, SDAP1 and SDAP2 in GM treated HEK-293 cells; (B) expression of p-PI3K/PI3K was determined by western blotting; (C) expression of p-AKT/AKT was determined by western blotting. Data are means ± SD (n = 3). **P < 0.01 as compared to the Control group. ##P < 0.01 as compared to the GM-treated group.

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Discussion

The clinical application of gentamicin is seriously limited by its ototoxic nephrotoxicity, which leads to the decrease of its antibacterial activity21. Although the specific mechanism of GM-induced nephrotoxicity remains unclear, there is increasing evidence that oxidative stress, inflammatory response and cell apoptosis play an important role in GM-induced nephrotoxicity22. Antioxidant therapy can help prevent this nephrotoxic effect23. The protein content of Sika deer antler is high (51% to 57%), which is considered to be the active ingredient to play a pharmacological role24. In addition, protein is the main carrier of life activities and plays a vital role in the smooth progress of various life processes. Therefore, the study of active proteins in pilose antler will be helpful to prevent nephrotoxicity and elucidate its molecular mechanism.

In a mouse model, we demonstrated that co-administration of SDAP can reduce GM-induced nephrotoxicity, as shown by the reduction in BUN, serum creatinine, and caspase-dependent apoptosis levels (Fig. 6). Increased ROS production is an important mechanism for GM-induced nephrotoxicity. Excessive levels of ROS can damage DNA, lipids and proteins, eventually leading to apoptosis25. SDAP has been shown to interact with ROS, thereby preventing ROS-induced tissue and cell damage. Consistent with previous studies, we found that GM-induced nephrotoxicity is significantly associated with increased MDA levels, increased LDH levels and decreased levels of antioxidant enzymes CAT, SOD and GSH26. In this study, SDAP treatment restored the levels of all these biomarkers to the normal range observed in the untreated control group (Fig. 8), confirming its protective effect on the kidneys. Histopathological examination of the mouse kidney also confirmed this protection (Fig. 7). Compared with the GM model group, the SDAP-treated group had significantly improved renal tubular necrosis and markedly relieved renal tissue damage. Mitochondria is the main target of oxidative damage caused by ROS27. In this study, GM treatment significantly disrupted the mitochondrial membrane potential (Fig. 10) of HEK293 cells, which is an indicative feature of mitochondrial dysfunction. Mitochondrion is the integration of internal and external apoptotic signals, which plays an important role in the initiation of mitochondrion mediated apoptosis28. The damaged mitochondrial membrane may affect the opening of the mitochondrial permeability transition pore (MPTP), which then leads to the release of cytochrome c and the activation of caspase-9, -3 and the formation of apoptotic bodies29. Caspase-3 is a key biomarker of apoptosis, which can be activated through internal and external apoptosis pathways, leading to DNA breakdown30. Apoptosis is mainly regulated by Bcl-2 family proteins, especially pro-apoptotic Bax and anti-apoptotic Bcl-2 proteins31. When Bax forms a heterodimer with Bcl-2, the release of cytochrome C from mitochondria and subsequent cell death are prevented32. In this study, activation of cleaved-caspase-3 was detected after GM exposure (Fig. 13). And 3 mg/mL GM could significantly increase the protein levels of Bax, and decrease the expression of Bcl-2 (Fig. 13). Interestingly, SDAP treatment significantly attenuated mitochondrial membrane destruction and the activation of cleaved-caspase-3, which subsequently reduced GM-induced apoptosis (Fig. 13). These results indicated that SDAP can reduce GM-induced apoptosis by inhibiting the mitochondrial apoptosis pathway.

Nrf2 is a key transcription factor that regulates antioxidant genes (such as CAT, SOD and HO-1) by combining with antioxidant response elements33. It has been shown that the activation of Nrf2 is related to cisplatin, acetaminophen and aristolochic acid in drug-induced nephrotoxicity34. Similarly, in HEK293 cells, after SDAP treatment, Nrf2 and its corresponding downstream HO-1, NQO1 protein levels increased significantly (Fig. 15). Consistently, SDAP pretreatment further activated Nrf2 and HO-1 protein expression, increased HO-1 activity, and then inhibited GM induced HEK293 apoptosis (Fig. 15). In addition, activated Nrf2 can induce the increase of GSH levels by inducing the expression of cysteine-glutamic acid exchange transporter, thereby protecting cells from oxidative stress35. These indicated that the activation of the Nrf2/HO-1 pathway may partly explain the activity of the antioxidant enzymes SOD and CAT and the SDAP-induced increase in GSH content. In conclusion, the activation of the Nrf2/HO-1 pathway plays a protective role in GM-induced apoptosis, and this activation also mediates the protective effect of SDAP.

NF-κB is a family of transcription factors, also known as major regulators in inflammation, oxidative stress and immunity36. NF-κB signaling pathway may be activated or inactivated by different factors or stimulation signals, such as tumor necrosis factor-A, IL-6, Caspase-3, c-Jun N-terminal kinase and ROS37. On the contrary, activation of NF-κB can induce the expression of many proinflammatory genes, including COX-2, TNF-α, IL-6 and iNOS38. Previous studies have shown that GM can induce severe inflammatory reactions, including activation of NF-κB and iNOS, and the production of TNF-α39. In this study, GM exposure significantly increased NF-kB protein expression and increased TNF-α and IL-6 levels (Fig. 17). Consistent with these findings, our results have shown that GM-induced nephrotoxicity is associated with significantly increased levels of NF-kB expression and TNF-α and IL-6 levels.

The Nrf2 and NF-κB systems may have crosstalk during oxidative stress and inflammation40. Nrf2 activation can inhibit the NF-κB nuclear translocation-mediated inflammatory response through HO-1 end products bilirubin and CO40. In this study, HO-1 activation (Fig. 15) attenuated GM-induced caspase-3 (Fig. 13) activation and NF-κB (Fig. 17) protein expression, and ultimately improved the protective effect of SDAP on GM-induced cytotoxicity. These indicated that Nrf2-mediated HO-1 activation partially contributes to SDAP's ability to inhibit the mitochondrial apoptosis pathway. Related research also shown that inhibiting HO-1 can enhance the expression of NF-κB and aggravate the inflammation induced by lipopolysaccharide (LPS)41. Therefore, SDAP can partially inhibit GM-induced inflammation through Nrf2/HO-1 activation and down-regulation of NF-κB expression. The inhibitory effect of SDAP on NF-κB may partially promote the activation of HO-1. Obviously, further research is needed to determine the detailed mechanism.

In short, in our study, SDAPR, SDAP1, and SDAP2 significantly improved GM-induced cytotoxicity of HEK293, mainly by improving inflammatory response, reducing apoptosis and enhancing antioxidant capacity. Further analysis indicated that SDAPR, SDAP1 and SDAP2 may play a chemopreventive role by activating Nrf2 and inhibiting NF-κB signaling pathway.

Conclusion

In conclusion, the results show that SDAP can relieve GM-induced nephrotoxicity by activating Nrf2 pathway. In addition, we found that SDAPR, SDAP1 and SDAP2 inhibited TNF-α expression, activation of NF-κB pathway and GM-induced inflammation in HEK293 cells. Furthermore, SDAP1 and SDAP2 enhanced Nrf2 activation in kidney tissue of GM-treated mice. In summary, our study suggests that SDAPR, SDAP1 and SDAP2, as natural antioxidants, may be used to treat human renal toxicity induced by GM.