Nephroprotective effect of magnesium isoglycyrrhizinate against arsenic trioxide-induced acute kidney damage in mice.

Magnesium isoglycyrrhizinate (MgIG) has anti-inflammatory, antioxidative, antiviral and anti-hepatotoxic effects. However, protective effects of MgIG against renal damage caused by arsenic trioxide (ATO) have not been reported. The present study aimed to clarify the protective function of MgIG on kidney damaged induced by ATO. Other than the control group and the group treated with MgIG alone, mice were injected intraperitoneally with ATO (5 mg/kg/day) for 7 days to establish a mouse model of kidney damage. On the 8th day, blood and kidney tissue were collected and the inflammatory factors and antioxidants levels in the kidney tissue and serum were measured. The expression of protein levels of caspase-3, Bcl-2, Bax, Toll-like receptor-4 (TLR4) and nuclear factor-κB (NF-κB) were determined via western blot analysis. In the renal tissue of mice, ATO exposure dramatically elevated markers of oxidative stress, apoptosis and inflammation. However, MgIG could also restore the activities of urea nitrogen and creatinine to normal levels, decrease the malondialdehyde level and reactive oxygen species formation and increase superoxide dismutase, catalase and glutathione activities. MgIG also ameliorated the morphological abnormalities generated by ATO, reduced inflammation and apoptosis and inhibited the TLR4/NF-κB signaling pathway. In conclusion, MgIG may mitigate ATO-induced kidney damage by decreasing apoptosis, oxidative stress and inflammation and its mechanism may be connected to the inhibition of TLR4/NF-κB signaling. Copyright: © Wei et al.

Introduction

Throughout history, arsenic has been recognized as both an environmental hazard and a therapeutic chemical (1). Arsenic trioxide (ATO) is widely considered an effective anti-cancer treatment medicine (2) and it is the leading treatment medicine for acute promyelocytic leukemia. Human populations exposed to high level of arsenic are at risk of developing skin, bladder, liver and lung cancers (3). Despite the well-known toxicity of ATO, it has been used to treat various diseases for centuries (4).

The kidney is an important organ that not only can maintain blood pressure and eliminate waste, but also can maintain and regulate body fluid, such as acid-base balance (5). Kidney damage or nephrotoxicity leads to the impairment of detoxification and excretion functions, which can be confirmed by renal markers such as blood urea nitrogen (BUN), creatinine (CRE) and CRE clearance (6).

Research has shown that ATO can cause kidney damage. Indeed, oxidation, inflammation and apoptosis have all been linked to ATO-induced nephrotoxicity (7,8). The primary mechanism by which ATO causes nephrotoxicity is oxidative stress (9). Additionally, oxidative stress can increase reactive oxygen species (ROS) production and malondialdehyde (MDA) level while decreasing superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) activities, leading to apoptosis (10,11). Thus, antioxidative agents may mitigate or prevent ATO-induced nephrotoxicity.

Toll-like receptor-4 (TLR4) acts as a lipopolysaccharide sensor to induce inflammation by activating factors that induce inflammation (12). TLR4 may activate the intercellular nuclear factor-κB (NF-κB), given the relationship between innate and acquired immunity (13). The NF-κB pathway is further activated, which leads to the entry of the p-p65 protein into the nucleus and increases the expressions of the pro-inflammatory factors (14,15).

ATO is capable of stimulating the formation of ROS (16). Overproduction of ROS is thought to activate the NF-κB pathway and result in the upregulation of pro-inflammatory mediators (17,18). On the whole, increased oxidative stress and consequent inflammation may result in a greater number of cells undergoing apoptosis (19). NF-κB is implicated in regulating numerous inflammatory genes and ATO-induced various stimuli can stimulate NF-κB (20). In conclusion, oxidative stress is recognized to serve a significant role in ATO-induced toxicity (21).

Magnesium isoglycyrrhizinate (MgIG; Fig. 1) is derived from 18-glycyrrhizic acid, which is a major component of natural licorice (Glycyrrhiza glabra L.) and has been shown to protect the liver and possesses anti-inflammatory, anti-oxidative and anti-apoptotic properties (22,23). In pharmacological experiments, MgIG has been demonstrated to suppress the inflammatory process, decrease pathological damage to hepatocytes and enhance the overall function of hepatocytes (24). Furthermore, MgIG protects the heart by inhibiting the TLR4/NF-κB signaling pathway, which is responsible for activating inflammatory factors (25). Licorice and its active components have been found to be protective against kidney damage (26). Nevertheless, no studies to date have examined the possible effects and mechanisms of MgIG on the nephrotoxicity caused by ATO.

Figure 1

Chemical construction of magnesium isoglycyrrhizinate.

The present study constructed a mouse model of ATO-induced kidney injury and examined the effects of MgIG on renal morphological and renal function to investigate the effects and potential mechanisms of MgIG in ATO-treated mice. In addition, the regulation of MgIG in the TLR4/NF-κB signaling pathways was investigated.

Materials and methods

Chemicals and drugs

MgIG for injection was purchased from the Chia Tai Tianqing Pharmaceutical Group Co., Ltd. ATO parenteral solution was purchased from Beijing SL Pharmaceutical Co., Ltd. All additional analytical-grade reagents were acquired from MilliporeSigma.

Animals

A total of 50 male Kunming mice (weight: 22.0±2.0 g, age: 6-7 weeks) were acquired from the Hebei Medical University Center for Laboratory Animals. The mice were fed at a normative temperature (22±2˚C) and humidity (50±10%). The mice were fed a standard pellet diet and water ad libitum. Animal studies at Hebei Medical University of Chinese Medicine (Shijiazhuang, China) were conducted in compliance with the Animal Care and Ethical Committee (approval no. DWLL2020005) and the United Kingdom Animal (Scientific Procedures) Act 1986. The Reporting of In Vivo Experiments guidelines were used to guide the current study (27).

Experimental design

Male mice were randomly divided into 5 groups (n=10): The control group (CONT; saline i.p. 10 mg/kg/day), the ATO group (ATO; i.p. 5 mg/kg/day) (28,29), the MgIG only group (MgIG; i.p. 50 mg/kg/day), the low-dose MgIG group (L-MgIG; i.p. 25 mg/kg/day) (30,31) and the high-dose MgIG group (H-MgIG; i.p. 50 mg/kg/day). With the exception of the CONT and MgIG groups, all mice were injected i.p. with ATO (5 mg/kg/day) for a total of 7 days. All 50 mice were still alive during the experiment and the body weight and status of the mice were checked daily. The criteria of humane endpoints for euthanasia were body weight loss (>10%) and anorexia in contrast with the controls. Following the last administration, the urine of the mice in each group was collected, after which the mice were anesthetized with sodium pentobarbital (50 mg/kg) and blood was drawn from the eyeballs. Then, the mice were sacrificed by intraperitoneal injection of an overdose of sodium pentobarbital (200 mg/kg) (28,32). Animal death was confirmed by the loss of signs of life, such as toe pinch response, heartbeat and breathing. After mice were sacrificed, kidney tissues were rapidly collected for subsequent experiments.

Urine and serum chemistry analysis

Blood (3,500 x g; 10 min; 4˚C) and urine (1,500 x g; 5 min; 4˚C) samples were obtained by centrifugation. The supernatant was then collected into Doff tubes. The serum and urine were refrigerated at -20˚C until use. The expression activities of BUN (cat. no. C013-2-1; Jiancheng Institute of Bioengineering) and CRE (cat. no. C011-2-1; Jiancheng Institute of Bioengineering) were determined by using colorimetric assay. Creatinine clearance was calculated according to the equation: Creatinine clearance (µl/min/g body weight)=[urinary CRE(µmol/l) x urine volume(µl/min)/serum CRE(µmol/l) x body weight(g)] (33,34).

Histopathological analysis

The kidney samples of mice were bisected, trimmed and fixed in 10% formalin at room temperature for 48 h. The samples were dehydrated in graded ethanol concentrations and then embedded in paraffin. Paraffin sections (4-µm) were stained with hematoxylin for 5 min and eosin for 3 min at room temperature. Light microscopy (Leica DM4000B; Leica Microsystems GmbH) was used to examine histological alterations. Score 1, 2, 3, 4 and 5 represent the kidney injury area <10, 10-25, 25-50, 50-75 and >75%, respectively. The renal injury was assessed by scoring tubular cell swelling, cellular vacuolization and others under the light microscopy in ≥10 different horizons and the average score calculated.

Measurement of oxidative stress and antioxidant enzymes

The collected serum and kidney tissues were used to detect the activities of GSH, SOD, CAT, MDA. The expression activities of GSH (cat. no. A006-2-1; Jiancheng Institute of Bioengineering), SOD (cat. no. A001-3-2; Jiancheng Institute of Bioengineering), CAT (cat. no. A007-1-1; Jiancheng Institute of Bioengineering) and MDA (cat. no. A003-1-1; Jiancheng Institute of Bioengineering) were determined by using colorimetric assay.

Measurement of ROS levels in the kidney

Ethoxylation is commonly used as a method to monitor the production of reactive ROS in cells (35). Dihydroethidium (DHE; cat. no. G1045l Wuhan Servicebio Technology Co., Ltd.) fluorescence was used to evaluate ROS production in the kidney. The kidney specimens were flash-frozen in liquid nitrogen. Then, the freezing microtome (Cryotome E; Thermo Fisher Scientific, Inc.) was used to cut slices. Frozen slices (5-µm) were incubated for 5 min with a spontaneous fluorescence-quenching reagent. The slides were then rinsed for 10 min with flowing water and the DHE was then dropped into the indicated region and incubated for 30 min at 37˚C in the dark. After washing with PBS (pH 7.4) in a Rocker device, the slides were incubated in a dark location with a DAPI solution for 10 min at room temperature. A total of 10 microscopic fields in each section were examined in a blinded method. A fluorescence microscope (Nikon Eclipse C1; Nikon Corporation) was used to observe and capture images at x200 magnification.

Inflammatory cytokine analysis

The kidney samples were promptly snap-frozen at -196˚C in liquid nitrogen for standby use. Interleukin-6 (IL-6; cat. no. 88-7064-88; MultiSciences Biotech Co., Ltd.), interleukin-1β (IL-1β; cat. no. 88-7013-88; MultiSciences Biotech Co., Ltd.) and tumor necrosis factor alpha (TNF-α; cat. no. 88-7324-88; Thermo Fisher Scientific, Inc.) levels in the kidney were measured by relevant ELISA kits.

Bax, Bcl-2, Caspase-3, TLR4, NF-κB and p-NF-κB by western blotting analysis

RIPA lysis buffer (Wuhan Servicebio Technology Co., Ltd.) was used to decompose the kidney samples. Centrifugation (12,000 x g; 10 min; 4˚C) was used to collect the total protein in tissue that concentration was determined with the BCA kit (catalog no. G2026; Wuhan Servicebio Technology Co., Ltd.). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (10%) was used to separate equal amounts of protein (15 µg). The proteins were transferred to polyvinylidene difluoride membranes that were soaked for overnight at 37˚C in a blocking solution containing 5% skimmed milk. Then, the membranes were incubated with anti-Bcl-2 associated X protein (Bax; cat. no. GB11690, 1:1,000; Wuhan Servicebio Technology Co., Ltd.), anti-B-cell lymphoma 2 (Bcl-2; cat. no. PAA778Mu01, 1:1,000; Cloud-Clone Corp.), anti-caspase-3 (cat. no. 66470-2-lg, 1:1,000; ProteinTech Group, Inc.), anti-NF-κB (cat. no. GM1003, 1:2,000; Wuhan Servicebio Technology Co., Ltd.), anti-p-NF-κB (cat. no. GB11142-1, 1:2,000; Wuhan Servicebio Technology Co., Ltd.), anti-TLR4 (cat. no. GB11519, 1:1,000; Wuhan Servicebio Technology Co., Ltd.) and β-actin (cat. no. GB12001, 1:1,000; Wuhan Servicebio Technology Co., Ltd.) antibodies at 4˚C for 12 h. Afterwards, the membranes were rinsed three times with PBS. Membranes were incubated for 2 h at 37˚C in darkness with secondary antibodies of goat anti-mouse IgG-HRP (cat. no. GB23301; 1:3,000; Wuhan Servicebio Technology Co., Ltd.) or goat anti-rabbit IgG-HRP (cat. no. GB23303; 1:3,000; Wuhan Servicebio Technology Co., Ltd.) to detect the protein bands. After subjecting the membranes to a chemiluminescent substrate (ECL; catalog no. G2014; Wuhan Servicebio Technology Co., Ltd.) and to autoradiographic film exposure, the optical density value of the target band was analyzed using Alpha software (Alpha Innotech).

Statistical analysis

Origin Pro 9.1 (OriginLab Corp.) software was used to perform statistical analyses. Data are demonstrated as mean ± SEM and one-way analysis of variance (ANOVA) and Tukey's post hoc test were used to assess statistically significant differences between groups. Non-parametric data were analyzed by the Kruskal-Wallis test and the Dunn-Sidak post hoc test (36). P<0.05 was considered to indicate a statistically significant difference.

Results

Effects of MgIG on alterations in biochemical indices

In Fig. 2, the levels of serum BUN, serum CRE, urinary CRE and CRE clearance were measured to examine the effect of MgIG on ATO-induced nephrotoxicity. Serum BUN and CRE levels were notably augmented and urinary CRE and CRE clearance levels were reduced in the ATO group (P<0.01) compared with the CONT group. However, the serum BUN and CRE levels of the H-MgIG and L-MgIG groups were markedly decreased but urinary CRE and CRE clearance levels were augmented in contrast with the ATO group (P<0.05 or P<0.01). There was no significant difference in levels of serum BUN, serum CRE, urinary CRE and CRE clearance between the CONT group and the MgIG group (P>0.05). MgIG may have an effect of renal protection.

Figure 2

Effects of MgIG on levels of serum (A) BUN and (B) CRE and urine (C) CRE and (D) creatinine clearance. Values are demonstrated as mean ± SEM, n=10. **P<0.01 vs. CONT group; ##P<0.01, #P<0.05 vs. ATO group. MgIG, magnesium isoglycyrrhizinate; BUN, blood urea nitrogen; CRE, creatinine; CONT, control group; ATO, arsenic trioxide group.

Effects of MgIG on the histopathological changes

As shown in Fig. 3, light microscopy was used to visualize hematoxylin and eosin-stained kidney slices. No morphological changes were found in the kidney tissues of the CONT and MgIG groups. By contrast, the ATO group showed tubular cell swelling, interstitial edema, inflammatory cell infiltration in the renal interstitial tissue, glomeruli dilation and hyperemia, partial epithelial cell necrosis (P<0.01). The mice who received H-MgIG and L-MgIG treatment had reduced areas of necrosis and inflammatory infiltration (P<0.05 or P<0.01). In other words, treatment of MgIG may ameliorated ATO-induced kidney damage in a dose-dependent manner.

Figure 3

Effects of MgIG on histopathological variations in mice, as observed by hematoxylin and eosin staining [(A) magnification, x400; scale bar=50 µm]. The CONT group shows the standard structure of glomerular capillaries and tubular epithelium; the ATO group exhibits the inflammatory cell infiltration in the renal interstitial, glomeruli dilation and hyperemia; the MgIG treatment group shows a standard structure; and the H-MgIG and L-MgIG groups alleviate renal morphological alterations. (B) A boxplot was used to depict the kidney injury scores, in which the band inside the box represents the median, and the bottom and top of the box represent the lower and upper quartiles, respectively. The circle and star represent the largest and smallest data respectively. Values are demonstrated as median ± range, n=10. **P<0.01 vs. CONT group; ##P<0.01, #P<0.05 vs. ATO group. MgIG, magnesium isoglycyrrhizinate; CONT, control group; ATO, arsenic trioxide group; H-MgIG, high MgIG group (50 mg/kg/day); L-MgIG, low MgIG group (25 mg/kg/day).

Effects of MgIG on the expression levels of antioxidant enzymes

As shown in Figs. 4 and 5, the ATO group showed markedly decreased the activities of SOD, CAT and GSH in serum and kidney tissues (P<0.01) and the content of MDA was increased, in contrast with the CONT group. Compared with the ATO group, the activities of SOD, CAT and GSH in the groups of MgIG therapy were enhanced (P<0.05 or P<0.01) and the level of MDA was decreased (P<0.01). In contrast to the control group, mice that only received 50 mg/kg MgIG showed no prominent difference in the serum and kidney tissues expression levels of SOD, MDA, CAT and GSH. Therefore, MgIG treatment may relieve ATO-induced oxidative stress.

Figure 4

Effects of MgIG on the expression levels of oxidative stress markers (A) SOD, (B) CAT, (C) MDA and (D) GSH in serum. Values are demonstrated as mean ± SEM. n=10. **P<0.01 vs. CONT group; ##P<0.01, #P<0.05 vs. ATO group. MgIG, magnesium isoglycyrrhizinate; SOD, superoxide dismutase; CAT, catalase; MDA, malondialdehyde; GSH, glutathione; CONT, control group; ATO, arsenic trioxide group.

Figure 5

Effects of MgIG on the expression levels of oxidative stress markers (A) SOD, (B) CAT, (C) MDA and (D) GSH in renal tissues. Values are demonstrated as mean ± SEM. n=10. **P<0.01 vs. CONT group; ##P<0.01, #P<0.05 vs. ATO group. MgIG, magnesium isoglycyrrhizinate; SOD, superoxide dismutase; CAT, catalase; MDA, malondialdehyde; GSH, glutathione; CONT, control group; ATO, arsenic trioxide group.

Effects of MgIG on ROS production in ATO-treated mice

As seen in Fig. 6, the kidney slices of mice were stained with a fluorescent dye that identified ROS. The ATO group had substantially greater ROS generation than the CONT group (P<0.01), whereas no significant changes were found between the CONT group and the MgIG group. In contrast to the ATO group, the H-MgIG and L-MgIG groups had lower ROS generation (P<0.05 or P<0.01). Thus, MgIG treatment may reduce the ROS production caused by ATO.

Figure 6

Effects of MgIG on the ROS levels in mice [(A) x200 magnification, scale bar=100 µm]. (B) ROS-positive cell area percentages in each group are shown. Values are demonstrated as mean ± SEM. n=6 **P<0.01 vs. CONT group; ##P<0.01, #P<0.05 vs. ATO group. MgIG, magnesium isoglycyrrhizinate; ROS, reactive oxygen species; CONT, control group; ATO, arsenic trioxide group.

Effects of MgIG on IL-6, IL-1β and TNF-α expression

Compared with the CONT group, the ATO group showed increase in IL-6, IL-1β and TNF-α expressions, as shown in Fig. 7 (P<0.01). MgIG alone treated mice did not show significant differences when compared to the CONT group. Nevertheless, the levels of IL-6, IL-1β and TNF-α in the H-MgIG and L-MgIG groups were decreased compared with the ATO groups (P<0.01). MgIG may have inhibitory effects on ATO-induced inflammatory reaction.

Figure 7

Effects of MgIG on the expression levels in renal tissues of IL-6 (A), IL-1β (B) and TNF-α (C). Values are demonstrated as mean ± SEM. n=10. **P<0.01 vs. CONT group; ##P<0.01 vs. ATO group. MgIG, magnesium isoglycyrrhizinate; IL, interleukin; TNF, tumor necrosis factor; CONT, control group; ATO, arsenic trioxide group.

Effects of MgIG on caspase-3, Bcl-2 and Bax expression

In the ATO group, protein expression levels of caspase-3 and Bax were upregulated, while Bcl-2 protein expression was downregulated, compared with the CONT group as show in Fig. 8 (P<0.01). However, the expression levels of caspase-3 and Bax were decreased and Bcl-2 expression level was significantly elevated in the L-MgIG group and H-MgIG group compared with the ATO group (P<0.05 or P<0.01). The expression levels of apoptotic proteins in MgIG alone treated mice did not show significant differences when compared to the CONT group. Thus, MgIG may alleviate the increase in the expression of pro-apoptosis indicators induced by ATO.

Figure 8

Effects of MgIG on the expression levels of apoptosis indexes were determined by western blot analysis. (A) For each group, the fluorescence intensity of (B) caspase-3, (C) Bcl-2 and (D) Bax was analyzed by normalizing to β-actin. Values are demonstrated as mean ± SEM. n=3. **P<0.01 vs. CONT group; ##P<0.01, #P<0.05 vs. ATO group. MgIG, magnesium isoglycyrrhizinate; Bcl-2, B-cell lymphoma 2; Bax, Bcl-2 associated X; CONT, control group; ATO, arsenic trioxide group.

Effects of MgIG on TLR4/NF-κB expression

As shown in Fig. 9, compared with the CONT group, the expressions of TLR4, p-NF-κB, NF-κB and p-NF-κB/NF-κB in the ATO group were markedly higher (P<0.01). TLR4, p-NF-κB, NF-κB and p-NF-κB/NF-κB expression levels in the H-MgIG and L-MgIG groups were lower compared with ATO group (P<0.05 or P<0.01). The expressions of TLR4, p-NF-κB, NF-κB and p-NF-κB/NF-κB in the MgIG group and CONT group demonstrated no significant difference.

Figure 9

Effects of MgIG on the expression levels of TLR4, NF-κB (p65) and p-NF-κB (p-p65). (A and D) For each group, the intensity of (B) TLR4, (C) NF-κB (p65), (E) p-NF-κB (p-p65) and (F) p-p65/p65 were analyzed by normalizing to β-actin. Values are demonstrated as mean ± SEM. n=3. **P<0.01 vs. CONT group; ##P<0.01, #P<0.05 vs. ATO group. MgIG, magnesium isoglycyrrhizinate; TLR4, Toll-like receptor-4; NF-κB, nuclear factor-κB; p-, phosphorylated; CONT, control group; ATO, arsenic trioxide group.

Discussion

Arsenic, a trivalent inorganic arsenic, also is a worldwide environmental pollutant and a human carcinogen (6,37,38). As MgIG has good lipophilicity, it easily penetrates the cell membrane to bind to the receptor protein and the target cell receptor of steroid hormones, thereby exerting hormonal effects (such as remission lipid metabolism disorder) (24,39).

The present study demonstrated that MgIG can ameliorate ATO-induced kidney damage in mice. In addition to modifying the ATO-mediated increase in BUN and CRE, oxidative stress and inflammatory cytokines were alleviated following MgIG treatment. The hematoxylin and eosin-stained kidney tissues of ATO-treated mice revealed abnormal kidney morphology and the kidney tissues of medication treatment groups showed slight anomaly, which indicted MgIG may possess the protective effect on kidney injury.

The kidney is the main organ of body used for arsenic excretion and also a principal site for arsenic accumulation. The present study found that MgIG could improve the pathological damage of the kidney and mitigate the abnormal variation of serum BUN, serum CRE, urinary CRE and CRE clearance. These markers are typically end-products of nitrogenous compounds and protein metabolism and they are used as biochemical indicators for detecting renal function (19,40,41). The considerable restoration of serum BUN, serum CRE, urinary CRE and CRE clearance activity levels showed that MgIG protected the kidney from the damage from ATO. However, the content of serum BUN, serum CRE, urinary CRE and CRE clearance in MgIG alone administered mice did not change abnormally. In conclusion, treatment of MgIG could alleviate ATO-induced nephrotoxicity.

Studies report that one of the mechanisms of nephrotoxicity induced by ATO is the excessive production of oxidative stress (42). In addition, its anticancer effects are also associated with abnormal oxidative stress (43). One of the ways to mitigate the oxidative stress caused by superfluous ROS is to augment the synergistic effects of antioxidant enzymes (GSH, SOD and CAT) (44). This is consistent with the conclusion of the present study that MgIG enhanced the activities of antioxidant enzymes and decreased the content of MDA and the production of ROS. In addition, the activities of GSH, SOD and CAT and the production of ROS and MDA in the MgIG group showed no significant change compared with those of the CONT group. As ATO can stimulate ROS production during the process of metabolic activation and ROS can devastate the structure of cells, it causes significant inflammation and ultimately leads to cell apoptosis (45). Therefore, ATO can further exacerbate nephrotoxicity by inducing oxidative stress and inflammation (46). The present study showed that the ATO group had the highest ROS content, which generated kidney damage. ROS can also activate all types of signaling pathways (such as TLR4/NF-κB pathway) (18).

Pro-inflammatory cytokines serve a significant position in the pathogenesis of various inflammatory illnesses (47). TNF-α is involved in cisplatin-induced nephrotoxicity (48). The activation of NF-κB can stimulate the transcription of IL-6, IL-1β and TNF-α genes, thereby aggravating the inflammatory response (47,49). The present study found that the levels of IL-6, IL-1β and TNF-α in renal tissues were considerably elevated in the ATO group compared with the CONT group, while treatment with MgIG lessened the expressions of inflammatory factors, illustrating the anti-inflammatory effect of MgIG (50,51).

Caspases and Bcl-2 family proteins (such as Bcl-2-like proteins, Bax-like proteins) are regulators of apoptotic signaling pathway (52). Bcl-2 is a membrane protein with anti-apoptotic effect and can restrain the activation of caspase-3. Bax is a death-promoting molecule and induces apoptosis (53). In the present study the ATO group demonstrated increased expression levels of caspase-3 and Bax and a decreased level of Bcl-2. However, the expression of apoptotic protein did not significantly change in the mice treated with MgIG alone. Thus, MgIG treatment slowed down the ATO-induced apoptosis by restoring the caspase-3, Bcl-2 and Bax expression to normal levels.

The activation of TLR can initiate the congenital inflammatory response as TLR4 can bind to a variety of ligands to trigger an inflammatory response (54,55). NF-κB is a downstream effector of the TLR4 signaling pathway and mediates a variety of inflammatory processes (56). In addition, activation of the NF-κB pathway leads to enhancement of p-p65 and increased expressions of IL-6, IL-1β and TNF-α, which eventually leads to the generation of inflammatory reactions in kidney tissue (57). ATO caused a notably increase the expression of NF-κB in kidney tissue. However, the mice treated with MgIG did not show a significantly change in the expression of TLR4, p-p65 and p65. Nevertheless, treatment with MgIG downregulated the TLR4 and NF-κB expression and inhibited the production of pro-inflammatory cytokines. Thus, the protective effect of MgIG in mice with acute renal injury may be due to its inhibition of TLR4 and NF-κB signaling.

In summary, the present study found that MgIG may protect against kidney toxicity through its antioxidant, anti-inflammation and anti-apoptosis properties. The results demonstrated that MgIG significantly mitigated ATO-induced nephrotoxicity by decreasing oxidative stress and inflammation, possibly via restraining the TLR4/NF-κB signaling pathway (Fig. 10). The results indicated that MgIG could be an effective therapy against ATO-induced nephrotoxicity. However, in clinical practice, the combination of MgIG with ATO requires further research.

Figure 10

Mechanism of MgIG on ATO-treated nephrotoxicity. MgIG, magnesium isoglycyrrhizinate; ATO, arsenic trioxide; TLR4, Toll-like receptor-4; BUN, blood urea nitrogen; CRE, creatinine; NF-κB, nuclear factor-κB; ROS, reactive oxygen species; IL, interleukin; TNF, tumor necrosis factor; CAT, catalase; GSH, glutathione; SOD, superoxide dismutase; MDA, malondialdehyde; Bax, Bcl-2 associated X; Bcl-2, B-cell lymphoma 2.