2019 Volume 44 Issue 3 Pages 167-175
The aim of this study was to explore the role of the NOD-like receptor family, pyrin domain containing (NLRP3) inflammasome and autophagy in Astragaloside IV (AS IV)-mediated protection against cisplatin-induced liver and kidney injury in rats. Rats were intraperitoneally administered cisplatin at a dose of 15 mg/kg and orally administered AS IV for 7 days. Histopathological and biochemical analysis were used to assess liver and kidney function. The levels and localization of NLRP3 and autophagy-associated protein were determined by Western blot and immunohistochemistry. Intraperitoneal administration of cisplatin induced acute liver and kidney injury, and activated the NLRP3 inflammasome. Oral administration of AS IV for 7 days protected against the cisplatin-induced injury, and inhibited the expression of NLRP3, as well as the production of pro-inflammatory cytokines. Moreover, cisplatin modulated the conversion of LC3 II and the expression of p62, thereby inhibiting autophagy and the activation of NLRP3. AS IV effectively protected against cisplatin-induced injury by inducing autophagy and limiting the expression of NLRP3. Autophagy-mediated NLRP3 inhibition might play a crucial role in AS IV-mediated protection against cisplatin-induced toxicity. These results provide evidence of a novel therapeutic that may be used to alleviate the toxic effects of platinum-based chemotherapy.
Cis-diamminedichloroplatinum (cisplatin) is an effective chemotherapeutic drug that is used to treat a wide range of cancers, such as non-small cell lung carcinoma, ovarian cancer, and testicular cancer (Zhou et al., 2017). However, the adverse events associated with cisplatin treatment, which occur in 28% to 36% of patients, limit its efficacy, many times resulting in the prolongation of treatment cycles. The kidneys and the liver are two of the organs where cisplatin accumulates, frequently leading to nephrotoxicity and hepatotoxicity (Gao et al., 2017; Isoda et al., 2018; K v et al., 2016). Moreover, the toxicity profile of cisplatin significantly reduces patient survival and quality of life. Presently, the specific mechanisms of cisplatin-induced nephrotoxicity and hepatotoxicity are not fully understood. An increase in inflammatory cytokines and the initiation of inflammatory responses have been reported as key factors in cisplatin-induced toxicity, though these exact mechanistic targets remain elusive (Benedetti et al., 2013; Rehman et al., 2014; Yan et al., 2017).
The NOD-like receptor family, pyrin domain containing (NLRP3) inflammasome is a multi-protein complex that comprises NLRP3, adaptor apoptosis-associated speck-like protein (ASC), and pro-caspase-1. It plays a key role in the release of several pro-inflammatory cytokines and chemokines, such as interleukin (IL)-1β and IL-18 (Hao et al., 2017; Shen et al., 2018; Song et al., 2016). In resting conditions, the expression of NLRP3 is relatively low. However, the expression of NLRP3 can be stimulated by many factors, including various toxicants and environmental irritants. Once NLRP3 is activated, the NLRP3 inflammasome is assembled, and pro-caspase-1 is autolytically cleaved to form activated caspase-1. Activated caspase-1 cleaves pro-IL-1β and pro-IL-18, thus allowing them to be secreted and inducing an inflammatory response (Elliott and Sutterwala, 2015; Pavillard et al., 2018; Vanaja et al., 2015). The activation of NLRP3 therefore functions to mediate inflammation and may play a key role in exacerbating cisplatin-induced toxicity.
Several inducers of NLRP3 activation have been previously identified (Abais, et al., 2015; Ding et al., 2018), but the inhibitiors of NLRP3 was rarely reported. Autophagy is involved in the removal of degraded proteins, damaged organelles, and other cytoplasmic constituents, and has been implicated in the inhibition of NLRP3 expression recently (Xu et al., 2018; Zhong et al., 2016a). Autophagy can regulate the assembly of the NLRP3 inflammasome by preventing the accumulation of damaged mitochondria, suggesting a pivotal role for autophagy in modulating inflammatory responses (Han et al., 2016; Jessop et al., 2016; Jiang et al., 2017; Spalinger et al., 2017; Zhong et al., 2016b). Therefore, autophagy activator may be an effective drug in preventing and treating cisplatin-induced inflammatory response.
Astragaloside IV (AS IV) is an active compound of the Traditional Chinese Herb Astragalus membranaceus, and has been shown to have broad cytoprotective effects, including hepatoprotective and renoprotective activity (Li et al., 2017). For example, AS IV inhibited the onset of diabetic nephropathy and renal fibrosis in vivo (Chen et al., 2014; Zhou et al., 2017). Yan et al. reported in 2017 that AS IV could protect against cisplatin-induced injury by inhibiting the activation of NF-κB and decreasing the levels of pro-inflammatory cytokines, including TNF-α and IL-Iβ (Yan et al., 2017). However, the specific mechanistic targets of AS IV are unknown.
In this study, we hypothesized that the activation of NLRP3 plays a key role in cisplatin-induced liver and kidney injury in rats, and that AS IV can protect against these effects via autophagy-mediated inhibition of NLRP3.
Specific pathogen-free male Sprague-Dawley (SD) rats (220-250 g each) were purchased from the Experimental Animal Centre of Jilin University, and all procedures were approved by the Animal Experiments Committee of the First Hospital of Jilin University. The rats were housed in a controlled environment (20-22°C, 55%-60% humidity, and 12-hr light/dark cycles), and fed with water and chow diet.
Cisplatin was purchased from Jiangsu Haosen Co. (Lianyungang, China). The antibodies used in this study included rabbit anti-NLRP3 (Bioss Antibodies, Beijing, China), rabbit anti-caspase-1 (Abcam, Cambridge, MA, USA), mouse anti-IL-1β (Santa Cruz, CA, USA), rabbit anti-LC3 (CST, Beverly, MA, USA), mouse anti-SQSTM1/p62 (Santa Cruz, CA, USA), and rabbit anti-β-actin (CST).
Forty male SD rats were randomly divided into four groups: (1) rats in the control group were intraperitoneally injected with vehicle (0.9% NaCl solution), and orally gavaged with water; (2) rats in the cisplatin group were intraperitoneally injected with cisplatin solution at a dose of 15 mg/kg, and orally gavaged with water; (3) rats in the low- or high-dose treatment group were intraperitoneally injected with cisplatin solution at a dose of 15 mg/kg, and orally gavaged with AS IV at a dose of 40 mg/kg or 80 mg/kg. AS IV or water were orally administered for 7 days, and, on the 4th day, cisplatin or vehicle was injected. Seventy-two hours after the administration of cisplatin or vehicle, the rats were anesthetized with chloral hydrate, blood was collected from the abdominal aortic puncture, and the rats were euthanized. Serum was obtained for biochemical analysis after centrifugation at 3000 rpm and stored at -20°C. Rat kidneys and livers were fixed in 4% formaldehyde for hematoxylin and eosin (HE) staining and immunohistochemical analysis. All other tissues were stored at -80°C for western blot analysis.
Rat body weight was measured prior to euthanasia. After euthanasia, livers and kidneys were surgically removed, cleaned with filter paper, and weighed. Liver/body and kidney/body weight index were calculated as (liver or kidney weight/body weight) × 100%.
Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), creatinine (Cr), and blood urea nitrogen (BUN) were detected with analysis kits purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China).
Rat livers and kidneys were fixed in 4% formaldehyde for 3 days, and embedded in paraffin. Liver and kidney sections (5 μm thick) were stained with HE and examined with a BX51 light microscope using a magnification of 400 times (OLYMPUS, Tokyo, Japan). An independent researcher blindly assessed the extent of liver and kidney injury in each section.
Rat liver and kidney sections were deparaffinized, rehydrated, and immunohistochemically stained using the DAB method. The slides were incubated overnight at 4°C with primary antibodies against NLRP3 (1:400), SQSTM1/p62 (1:100), and then labeled with streptavidin-peroxidase (SP). The immunoreactive proteins were viewed with a BX51 light microscope (OLYMPUS), and blindly analyzed by an independent reviewer at a magnification of 400 times.
Rat livers and kidneys were homogenized in RIPA buffer, and centrifuged at a speed of 12,000 rpm for 10 min at 4°C. Proteins diluted with 20% loading buffer were loaded onto precast SDS-PAGE gels, then transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk in Tris-buffered saline (pH 7.5) with 0.1% Tween-20 (TBST) for 1 hr at room temperature. The membranes were then incubated overnight at 4°C with rabbit anti-NLRP3 (1:500), rabbit anti-capase-1 (1:1000), mouse anti-IL-1β (1:200), rabbit anti-SQSTM1/p62 (1:500), rabbit anti-LC3 (1:1000) and rabbit anti-β-actin (1:2000). The membranes were washed for 10 min three times with TBST, and incubated with secondary antibodies for 1 hr at room temperature. Immunoreactive proteins were visualized using an ECL kit (GE Healthcare, Amersham, UK), and the density of the bands was analyzed using Image J software (Bethesda, MD, USA).
All data are presented as means ± standard deviation (SD) and were analyzed by SPSS 18.0 software. Differences between groups were determined using one-way ANOVA followed by the Tukey test when equal variances were assumed. Differences between groups were considered to be statistically significant when p < 0.05.
Our assessment of overall survival, as shown in Fig. 1, shows that rats from the control group were all alive by day 8, and that the overall survival of rats treated with cisplatin was 50%. The survival of rats administered AS IV at doses of 40 and 80 mg/kg was 70% and 90%, respectively. Of the rats administered AS IV and cisplatin, those treated with a dose of 80 mg/kg had a slightly increased overall survival compared with those who were administered cisplatin alone. Although there was no significant difference between those treated with cisplatin alone and those co-treated with a dose of 80 mg/kg AS IV due to the limited numbers of rats, it also could indicate an overall protective effect of AS IV.
The overall survival of rats treated with cisplatin and AS IV. (n = 10).
Data in Fig. 2A show that the liver/body weight index was significantly increased in rats treated with cisplatin compared with the control group (p < 0.001). In response to the administration of AS IV, this index was found to be decreased at doses of 40 mg/kg (p < 0.01) and 80 mg/kg (p < 0.001). The high liver/body weight index in cisplatin-treated rats is suggestive of hepatotoxicity and liver swelling.
Assessment for liver injury of rats upon different treatments. (A) Effect on liver/body weight index. (B) Levels of ALT in rat serum. (C) Levels of AST in rat serum. (D) Images of rat liver histopathological sections (× 400). (1, necrotic foci; 2, acidophilic change of the hepatocytes). *p < 0.05, **p < 0.01 and ***p < 0.001. (n = 5).
In agreement, hepatic function, as assessed by ALT and AST levels in serum, was significantly increased in rats treated with cisplatin compared with the control group (p < 0.001; Fig. 2B and 2C), which indicated injury in rat liver after exposure of cisplatin. In rats dosed with 40 and 80 mg/kg of AS IV, ALT and AST levels were also found to be decreased compared with the cisplatin-treated group (p < 0.05), and were also found to have a more significant therapeutic effect in the high-dose group. This demonstrates that AS IV could alleviate cisplatin-induced liver injury in a dose-dependent manner.
Furthermore, we found that cisplatin induced necrotic foci in hepatocytes. In rats that were treated with both AS IV (40 mg/kg) and cisplatin, only acidophilic changes were observed. Livers from the control group and the AS IV group (80 mg/kg) had normal and eumorphic hepatocytes (Fig. 2D). Overall, administration of AS IV appeared to ameliorate cisplatin-induced liver injury in rats, with a more pronounced effect at a high dose of AS IV.
Data in Fig. 3A also show that the kidney/body weight index was significantly increased in rats treated with cisplatin compared with the control group (p < 0.001), while the kidney/body weight index of rats treated with either 40 or 80 mg/kg of AS IV was reduced (p < 0.05 and p < 0.01, respectively).
Assessment for kidney injury of rats upon different treatments. (A) Effect on kidney/body weight index. (B) Levels of creatinine in rat serum. (C) Levels of blood urea nitrogen in rat serum. (D) Images of rat kidney histopathological sections (× 400). (1, vacuolization and swelling; 2, protein cast). *p < 0.05, **p < 0.01 and ***p < 0.001. (n = 5).
The levels of Cr and BUN were also significantly increased in the cisplatin group compared with the control group (p < 0.001), and administration of either 40 or 80 mg/kg of AS IV resulted in lower levels of Cr (p < 0.01 and p < 0.001, respectively, Fig. 3B) and BUN (p < 0.001, respectively, Fig. 3C).
Following the light microscopy assessment at × 400 magnification (Fig. 3D), our histopathological assessment of the kidneys revealed a high extent of injury in the cisplatin group. Samples from this group showed protein cast, cavitation, and swelling of renal tubular epithelial cells. In rats treated with 40 mg/kg of AS IV, only protein cast was observed, while there were no signs of kidney injury in rats treated with 80 mg/kg of AS IV.
We found that the liver- and kidney-specific expression levels of NLRP3, IL-1β, and caspase-1 were significantly elevated in rats treated with cisplatin compared with the control group, an effect that was abrogated by administration of 40 and 80 mg/kg of AS IV, with a larger reduction in protein levels occurring in the 80 mg/kg group (Fig. 4A and 4B). NLRP3 was mainly localized to the cytoplasm of the hepatocytes and nephrocytes upon exposure to cisplatin, and NLRP3 expression in the kidneys was observed in both renal tubular and glomerulus cells (Fig. 4C). The expression levels were reduced in response to 40 and 80 mg/kg of AS IV, especially with less NLRP3 expression observed with 80 mg/kg.
Expression of NLRP3 in response to cisplatin and AS IV. (A) Expression levels of NLRP3, IL-1β, and caspase-1 in rat livers by western blot. (B) Expression of NLRP3, IL-1β, and caspase-1 in rat kidneys by western blot. (C) Images of rat liver and kidney immunohistochemistry sections stained for NLRP3 expression (× 400). The relative expression of protein was calculated as band intensity of target protein normalized to β-actin. *p < 0.05, **p < 0.01, and ***p < 0.001. (n = 4).
We found that the liver- and kidney-specific expression levels of LC3 II/I and p62 were significantly decreased and increased, respectively, in response to cisplatin compared with the control group, indicating a possible inhibition of autophagy, an effect that was reversed by administration of 40 and 80 mg/kg AS IV, with a more pronounced effect in the 80 mg/kg group (Fig. 5A, 5B and 5C). These results suggest that AS IV can induce autophagy, thereby protecting the rats from cisplatin-mediated liver and kidney injury.
Expression levels of LC 3 II/I and p62 in response to cisplatin and AS IV. (A) Expression levels of LC3 II/I and p62 in rat livers, as measured by western blot. (B) Expression of LC3 II/I and p62 in rat kidneys, as measured by western blot. (C) Images of rat liver and kidney immunohistochemistry sections stained for p62 expression (× 400). The relative conversion of LC3 II/I was calculated as the band intensity of LC3 II normalized to LC3 I. The relative expression of p62 was calculated as the band intensity of p62 normalized to β-actin. *p < 0.05, **p < 0.01, and ***p < 0.001 versus the control group. (n = 4).
Cisplatin treatment is commonly associated with serious adverse effects, such as severe acute liver and kidney injury, limiting its clinical use (Gong et al., 2015). In this study, we demonstrated that AS IV protects against cisplatin-induced acute liver and kidney injury in rats through inhibiting the expression of NLRP3 and the assembly of the NLRP3 inflammasome. Furthermore, we showed that the inhibition of NLRP3 is likely due to the activation of autophagy (Fig. 6). The link between cisplatin-induced NLRP3 activation and autophagy has not previously been identified, and may provide new insights into the mechanisms of cytoprotection of Traditional Chinese Herbs in acute liver and kidney injury.
Schematic diagram representing the mechanism of AS IV-mediated protection in cisplatin-induced injury. In this model, autophagy inhibition increases the number of damaged mitochondria and triggers the expression of NLRP3, thereby promoting the assembly of the NLRP3 inflammasome. NLRP3 cleaves pro-caspase-1, thus leading to the accumulation of active caspase-1, which further cleaves pro-IL-1β and pro-IL-18, resulting in the production of mature pro-inflammatory cytokines (IL-1β and IL-18). These cytokines then induce an inflammatory response. Based on our data, cisplatin activates the NLRP3 inflammasome by inhibiting autophagy, an effect that is reversed by treatment with AS IV.
Cisplatin-induced liver and kidney dysfunction is a multifactorial process involving inflammatory responses. Previous reports have indicated that an increase in the levels of pro-inflammatory cytokines, particularly IL-1β, is associated with cisplatin-induced kidney injury (Adams et al., 2010; Ozkok et al., 2016). The production of IL-1β is mediated by the assembly of the NLRP3 inflammasome. Our data suggest that cisplatin upregulates NLRP3, thereby elevating the levels of IL-1β in rat livers and kidneys. Additionally, this effect was abrogated by AS IV in a dose-dependent manner.
Damaged mitochondria can produce many harmful substances, including the stimulus of NLRP3. However, the damaged mitochondria could be removed by autophagy, a process that eliminates damaged proteins and organelles, thereby controlling such processes as inflammation and oxidative stress. Accumulating evidence suggests that autophagy inhibits the expression of NLRP3 and decreases the secretion of pro-inflammatory cytokines (Hu et al., 2018; Liu et al., 2018). Our data suggest that autophagy was induced by treatment with AS IV and this is similar to other studies that have shown that treatment with AS IV activates autophagy (Liu et al., 2017; Lu et al., 2015). Therefore, the autophagy activation might be an important mechanism in AS IV-mediated cytoprotection against cisplatin-induced liver and kidney injury.
In conclusion, our data demonstrate that AS IV is protective against cisplatin-induced liver and kidney injury by inhibiting the expression of NLRP3 and blocking the release of pro-inflammatory cytokines. Furthermore, we show that this effect might be associated with increased autophagy, which may regulate the expression and activity of NLRP3. Our data highlight the importance of these pathways in chemotherapy-induced liver and kidney injury, and provide a basis for targeting these pathways to alleviate the deleterious effects of toxic compounds, such as cisplatin, that are used to treat life-threatening diseases like cancer.
This work was supported by the National Natural Science Foundation of China under Grant (numbers 81803608); and the Administration of Traditional Chinese Medicine of Jilin Province, China under Grant (numbers 2018113).
The authors declare that there is no conflict of interest.