Aloperine Protects Mice against Ischemia-Reperfusion (IR)-Induced Renal Injury by Regulating PI3K/AKT/mTOR Signaling and AP-1 Activity
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Aloperine is a quinolizidine alkaloid extracted from the leaves of Sophora plants. It has been recognized with the potential to treat inflammatory and allergic diseases as well as tumors. In this report, we demonstrate that pretreatment with aloperine provided protection for mice against ischemia-reperfusion (IR)-induced acute renal injury as manifested by the attenuated inflammatory infiltration, reduced tubular apoptosis, and well-preserved renal function. Mechanistic studies revealed that aloperine selectively repressed IL-1β and IFN-γ expression by regulating PI3K/Akt/mTOR signaling and NF-κB transcriptional activity. However, aloperine did not show a perceptible impact on IL-6 and TGF-β expression and the related Jak2/Stat3 signaling. It was also noted that aloperine regulates AP-1 activity, through which it not only enhances SOD expression to increase reactive oxygen species (ROS) detoxification but also promotes the expression of antiapoptotic Bcl-2, thereby preventing tubular cells from IR-induced apoptosis. Collectively, our data suggest that administration of aloperine prior to IR insults, such as renal transplantation, could be a viable approach to prevent IR-induced injuries.
Renal ischemia and reperfusion (IR) injury is one of the major causes of acute kidney disease, a disorder with high morbidity and mortality in clinical settings (1). Indeed, IR inevitably resulted from the consequences of renal transplantation, aortic bypass surgery, and vascular surgery (2, 3, 4, 5, 6). There is overwhelming evidence that IR breaks the immune homeostasis by causing changes in endothelial cells, tubular epithelial cells, leukocytes, and renal parenchymal cells (7, 8, 9, 10, 11). Particularly, interplay between inflammatory cytokines/chemokines, apoptotic factors and reactive oxygen species (ROS) has been observed during the course of IR-induced renal injury (12, 13, 14, 15). Because of this complexity, its underlying molecular mechanisms remained poorly understood (16,17). It is believed that the innate immune system is activated in the very early stage of IR insult as manifested by the accumulation of neutrophils, dendritic cells (DCs) and macrophages, followed by the release of copious amounts of inflammatory mediators (18,19). Given that tubular cells are very susceptible to ROS (20, 21, 22), renal tubular apoptosis is a characteristic feature for the progression of IR insult (23).
Previous studies have consistently demonstrated that aloperine, an alkaloid extracted from sophora alopecuroides, possesses beneficial properties against inflammation, tumor growth and infection (24, 25, 26, 27, 28). As a result, it has been used widely in clinical settings for the treatment of inflammatory diseases including allergic dermatitis (29). More interestingly, recent studies further suggest the potential for aloperine to attenuate oxidative stress (30). These findings prompted us to hypothesize that aloperine could be a good candidate drug for the prevention and treatment of IR-induced acute renal injury. To address this feasibility, we conducted studies in mice with IR insult. Our results revealed that pretreatment with aloperine provided protection for mice against IR-induced acute renal injury as manifested by the attenuated inflammatory infiltration and reduced tubular apoptosis along with improved renal function. Our data suggest that administration of aloperine prior to IR insults could be a viable approach to the prevention of IR-induced renal injuries in clinical settings.
Materials and Methods
Male C57BL/six mice (7 wks old, body-weight 25–30 g) were purchased from the Animal Experimental Center of Hubei Province (Wuhan, China) and housed in a specific pathogen-free facility with free access to sterile acidified water and irradiated food. After a minimum acclimation of 1 wk, the mice were randomly allocated into four groups, each containing six mice: i) IR-aloperine group (IR + Alo) mice were pretreated with 50 mg/kg bodyweight of aloperine (Q1962, Kmaels) dissolved in saline containing 10% acetic acid (2.5 mg/mL), by gavage for 8 consecutive days before IR insult; ii) I/R-vehicle group (IR) mice were pretreated with 10% acetic acid in normal saline as above followed by IR insult; iii) Sham group (Sham) mice underwent similar surgical procedures but without IR insult; and iv) Sham-aloperine group (Sham + Alo) mice were administered with the same dose of aloperine as above. The aloperine dose (50 mg/kg) was optimized by pilot experiments, and no cytotoxicity was observed until the dosage reached 150 mg/kg for 8 consecutive days. All studies were conducted according to the NIH guidelines (Guide for the Care and Use of Laboratory Animals, 8th edition, 2011) and approved by the Institutional Animal Care and Use Committee at the Tongji Hospital, Huazhong University of Science and Technology.
RAW264.7 and HK2 cells were cultured in the complete RPMI-1640 medium containing 10% FBS and 1% antibiotics (100 IU/mL penicillin, 100 µg/mL streptomycin) at 37°C under 5% CO2 for 24 h, then subjected to serum starvation for 24 h by reducing FBS to 2%. After washes, the monolayer was immersed in mineral oil for 3 h, followed by culturing the cells under normal conditions for another 24 h as reported (31,32). HK2 and RAW264.7 cultures were divided into four groups: i) normal control (Nor) cells were cultured under normal condition; ii) hypoxic control (Hypo) cells were subjected to hypoxic insult; iii) hypoxia + aloperine (Hypo + Alo) cells underwent hypoxic insult in the presence of 0.5 mmol/L aloperine; and iv) hypoxia + aloperine + Tanshinone IIA (an inhibitor of for AP-1) (Hypo + Alo + TanIIA) cells were subjected to hypoxia in the presence of aloperine and Tanshinone IIA (TanIIA, 8 µg/mL).
Induction of Renal IR Injury
The mice were anesthetized by intraperitoneal (IP) injection of 1% pentobarbital (80 mg/kg bodyweight), and then placed in a prone position on a heating pad to maintain their body temperature during surgery. After bilateral flank 1-cm incisions, both kidneys were subjected to ischemia for 45 min using microvascular clamps (0.75-mm clip width and 5-mm jaw length, RoBoz Surgical Instrument Co.) and then reperfused as previously described (33). All mice were administered 200 µL of saline through subcutaneous injection to maintain fluid balance. After the surgery, the mice had access to food and water ad libitum. Mice were killed 24 h after reperfusion. Plasma samples and renal tissues were collected for experimental purposes.
Assessment of Renal Function
Renal function was assessed by measuring blood urea nitrogen (BUN) and serum creatinine (Cr) at the Department of Clinical Laboratories of Tongji Hospital.
Paraffin embedded renal sections (3 µm) were subjected to hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining as reported previously (34,35). Morphological changes in the cortex and medulla were scored in a blinded fashion by two pathologists according to the severity of tubular necrosis, integrity of brush border and basement membrane, and cast formation. The scoring included 4 scales: 0) <10%; 1) 10% to 25%; 2) 25% to 50%; 3) 50% to 75%; and 4) >75% (36). Each section was assessed with randomly selected 10 high-power fields (magnification, × 400), and three mice were included in each study group.
Myeloperoxidase (MPO) and F4/80 Immunostaining
Renal sections were deparaffinized and treated with proteinase K (Merck). The sections were first treated with 3% H2O2 to block endogenous peroxidase activity, followed by incubation with antibodies against F4/80 antibody (ABD) (Serotec Inc.) and MPO (Thermo Scientific), respectively. After washes with PBS, the sections were incubated with a biotinylated anti-rat IgG (ZSJQ-BIO). The sections were then developed using a Vector stain ABC kit (Vector Laboratories Inc.), and further counterstained with Harris hematoxylin as reported previously (37). Assessment of neutrophil and macrophage infiltration was carried out in a blinded fashion under a microscope by counting the MPO- or F4/80-positive cells per field with 10 randomly selected fields per section at magnification of 400×; three mice were examined in each study group.
Real-Time PCR Analysis
Renal total RNA extraction and cDNA synthesis were conducted using the established techniques (38). Real-time PCR was then carried out using an ABI prism 7500 Sequence Detection System (Applied Biosystems) with primers specific for IL-1β (5′-GGA TGA GGA CAT GAG CAC CT-3′, 5′-GGA GCC TGT AGT GCA GTT GT-3′), IL-10 (5′-CCC TTT GCT ATG GTG TCC TT-3′, 5′-TGG TTT CTC TTC CCA AGA CC-3′), IFN-γ (5′-GGC ACA GTC ATT GAA AGC CTA-3′, 5′-CTG CAG GAT TTT CAT GTC ACC-3′), IL-6 (5′-AGT TGC CTT CTT GGG ACT GA-3′, 5′-TCC ACG ATT TCC CAG AGA AC-3′), and TGF-β (5′-CAA CAA TTC CTG GCG TTA CCT TGG-3′, 5′-GAA AGC CCT GTA TTC CGT CTC CTT-3′). Glyceralhyde-3-phosphate dehydrogenase (GAPDH) was used for normalization (5′-TGG CAT TGT GGA AGG GCT CA-3′, 5′-GCA CCA GTG GAT GCA GGG AT-3′). PCR amplifications were carried out at 95°C for 1 min, followed by 40 cycles at 95°C for 15 sec, 60°C for 1 min. Relative expression levels for each target gene were calculated using the 2−ΔΔCt approach (38).
Assays for ROS Accumulation, MDA Level and SOD Activity
For analysis of ROS accumulation, fresh frozen sections were incubated with 2′ 7′-dichlorodihydrofluorescein diacetate (DCFH-DA, 10 µmol/L, YIJI) at 37°C for 25 min. After washes, the sections were subjected to fluorescence analysis under a fluorescence microscope (Olympus, Japan). The intensity of fluorescence on each section was analyzed using the Image Pro Plus software (Media Cybernetics) as reported (39). SOD activity and MDA levels were measured by the commercial kits from Nanjing Jiancheng Bioengineering Institute according to their respective instructions.
Western Blot Analysis
Cell or renal lysates (50 µg) were separated on 10% polyacrylamide gel (Sigma-Aldrich) and transferred onto PVDF membranes. The membranes were blocked with 5% nonfat milk for 1 h and then incubated with antibodies with optimal dilutions against PI3Kp85, p-PI3Kp85, AKT, p-AKT, mTOR, p-mTOR, Bcl2, c-Fos, Jak2, p-Jak2, Stat3, p-Stat3 (all Cell Signaling Technology), GAPDH, lamin B, caspase-3 and NF-κBp65 (all Santa Cruz Technologies) overnight at 4°C. After six washes (each for 5 min), the membranes were probed with an HRP-conjugated secondary antibody and then visualized using an ECL Plus Western blot kit (PIERCE) as reported previously (40). GAPDH was used for normalization, and the intensity of each reactive band was analyzed using the densitometry plugin ImageJ software (https://doi.org/rsb.info.nih.gov/ij/) as instructed.
Terminal Deoxynucleotidyl Transferase dUTP Conjugated with Fluorescein (TUNEL) Assay
Apoptotic cells in renal sections were estimated by TUNEL assay using a kit from Roche Diagnostics using the established techniques (41). Apoptotic rate was assessed by averaging the total number of apoptotic cells in 10 randomly selected fields at 400 × magnification of each section in a blinded manner. Three sections were examined for each mouse and three mice were included in each study group.
Electrophoretic Mobility Shift Assay (EMSA)
Nuclear proteins derived from HK2 cells and macrophages were prepared using the NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Scientific), and then subjected to EMSA using a LightShift Chemiluminescent EMSA Kit (Thermo Scientific) as described previously (34). Biotin-labeled double-stranded oligonucleotides containing the NF-κbp65 (biotin-5′-AGT TGA GGG GAC TTT CCC AGG C-3′, biotin-5′-GCC TGG GAA AGT CCC CTC AAC T-3′) or AP-1 binding site (biotin-5′-CGC TTG ATG ACT CAG CCG GAA-3′, biotin-5′-TTC CGG CTG AGT CAT CAA GCG-3′) (Beyotime) were used as the probe for analysis of NF-κB and AP-1 DNA binding activity, respectively. A mixture of unlabeled probes was used as a negative control.
All data are present as mean ± SEM, and all in vitro experiments were conducted with at least three independent replications. A P value of <0.05 was considered statistically significant. The GraphPad Prism 5 software was employed for statistical analysis using Student t test or one-way or two-way ANOVA where appropriate.
All supplementary materials are available online at https://doi.org/www.molmed.org .
Aloperine Provides Protection for Mice against IR-Induced Acute Renal Injury
Aloperine Pretreatment Attenuates IR-Induced Inflammatory Response
Administration of Aloperine Prevents IR-Induced Tubular Apoptosis
Aloperine Pretreatment Reduces IR-Induced ROS Accumulation
Aloperine Suppresses PI3K/Akt/mTOR Signaling and NF-κB Activity
To confirm the above results, we cultured RAW264.7 cells, a murine macrophage cell line, under hypoxic conditions in the presence or absence of aloperine as described, followed by analysis of NF-κB activity. We first conducted Western blot analysis to confirm the effect of aloperine on PI3K/Akt signaling in RAW264.7 cells. Indeed, aloperine significantly attenuated hypoxia-induced PI3Kp85 and Akt activation (Supplementary Figure 1D). Similarly, treatment of RAW264.7 cells with aloperine markedly inhibited hypoxia-induced NF-κB activation as evidenced by the reduced amount of p-p65 in the nucleus (Figure 6E). To further confirm these data, EMSA was employed to assess NF-κB DNA binding activity. It was noted that the addition of aloperine attenuated hypoxia-induced NF-κB DNA binding activity by almost one-fold (Figure 6F). Collectively, our data support that aloperine inhibits PI3K/AkT/mTOR signaling, thereby repressing NF-κB activity to attenuate IL-1β and IFN-γ expression.
Aloperine Enhances AP-1 Activity to Repress Tubular Apoptosis and to Promote IL-10 Secretion
Next, we cultured HK2 cells, a tubular cell line, under hypoxic conditions as above in the presence of aloperine along with the administration of Tanshinone IIA (TanIIA), an AP-1-inhibitor. Indeed, hypoxia induced a significant upregulation for nuclear c-Fos (Figure 7C) along with enhanced Bcl-2 expression (Figure 7D) as compared with that of cells cultured under normoxic condition. Importantly, significantly higher levels of c-Fos (Figure 7C) and Bcl-2 expression (Figure 7D) were noted in cells cultured with aloperine as compared with cells without aloperine, while the addition of TanIIA almost completely abolished the effect of aloperine. In line with these results, significantly higher AP-1 DNA binding activity was detected in aloperine-treated HK2 cells, which was abrogated by the addition of TanIIA (Figure 7E).
Because AP-1 also serves as a potent transcription factor for IL-10 expression (46), we then treated RAW264.7 cells with aloperine as above in the presence of TanIIA. As expected, aloperine stimulated RAW264.7 cells expression of high levels of IL-10, while blockade of AP-1 activity by TanIIA almost completely abolished aloperine-induced IL-10 expression (Figure 7F), indicating that aloperine enhances IL-10 expression by regulating AP-1 activity. The next important question is why administration of aloperine did not affect IL-6 and TGF-β expression. Given the role that Jak2/Stat3 signaling played in the regulation of IL-6 and TGF-β transcription (47), we conducted Western blot analysis to check the impact of aloperine on Jak2/Stat3 activity. As expected, IR insult induced a significant increase for total Jak2 and phosphorylated Jak2 (p-Jak2) (Figure 7G), and also for total Stat3 and phosphorylated Stat3 (p-Stat3) (Figure 7H). However, we failed to detect a perceptible difference between IR and IR + Alo mice. To confirm this observation, we further examined Jak2/Stat3 signaling in the above-prepared RAW264.7 and HK2 cells with hypoxic insult, and similar results were obtained (data not shown). Collectively, these data suggest that aloperine selectively regulates PI3K/Akt signaling and AP-1 activity, but does not affect Jak2/Stat3 signaling and, as a result, administration of aloperine did not result in a significant change for IL-6 and TGF-β expression.
Although aloperine has been recognized with therapeutic potential for inflammatory and allergic diseases in clinical settings (28,29), its effect on IR-induced acute renal injury, however, has yet to be elucidated. In this study, we induced mice with 45 min ischemia along with aloperine precondition followed by reperfusion, and IR injury was assessed 24 h after reperfusion. We demonstrated convincing evidence suggesting that pretreatment with aloperine provides protection for mice against IR-induced acute renal injury as manifested by the well preserved renal function along with attenuated pathological changes and inflammatory infiltration. Our data support that aloperine could be an alternative therapy for prevention of IR injury in clinical settings.
IL-1β, one of the most important proinflammatory cytokines, is generally produced at sites of tissue injury or infection by infiltrated immune cells (48). Similarly, IFN-γ is another potent proinflammatory cytokine playing a central role in inflammatory responses (49). By contrast, IL-10 possesses antiinflammatory properties in limiting host immune response to pathogens or tissue injury (50). Consistent with the role both IL-1β and IFN-γ played during the course of inflammatory responses, IR insult resulted in a significant upregulation of IL-1β and IFN-γ in the kidneys. However, pretreatment of mice with aloperine markedly attenuated renal IL-1β and IFN-γ expression, but the expression of IL-10 was increased by four-fold, indicating that aloperine protects mice against IR-induced injury by repressing inflammatory responses. Interestingly, unlike its effect on IL-1β and IFN-γ, we failed to obtain data supporting that aloperine modulates IL-6 and TGF-β expression.
The mechanisms underlying IR-induced acute renal injury are complex and have yet to be fully elucidated. However, MAP kinases and PI3K/Akt/mTOR signaling are thought to be implicated in the initiation and progression of IR-induced inflammatory responses (51, 52, 53, 54). We first examined the impact of aloperine on MAP kinase activities. Unexpectedly, we failed to detect a perceptible change in terms of the expression for total and activated (phosphorylated) ERK1/2, p38 and JNK, suggesting that MAP kinases are not relevant to an aloperine-mediated protective effect in our model. These results prompted us to embark on the PI3K/Akt/mTOR signaling, which is well known to regulate a variety of essential cellular functions ranging from glucose metabolism, protein synthesis, cell proliferation, apoptosis and survival (55, 56, 57, 58). PI3K consists of two elements, the regulatory subunit p85 and the catalytic subunit p110 (22,59). Its function relies on the activation of p85. Unlike its effect on MAP kinases, mice pretreated with aloperine manifested significantly attenuated levels for phosphorylated PI3Kp85, Akt and mTOR, while total PI3Kp85, Akt and mTOR were not changed. In line with these results, the phosphorylated levels for NF-κBp65 in the nucleus were reduced significantly in aloperine-pretreated mice as compared with that of IR mice. Indeed, RAW264.7 cells cultured under hypoxic conditions in the presence of aloperine manifested attenuated PI3K/Akt/mTOR signaling, which then repressed the activation of NF-κBp65 along with reduced DNA activity. In looking at this data together, our results suggest that aloperine attenuates inflammatory responses in the setting of IR-induced acute renal injury by repressing PI3K/Akt/mTOR signaling and NF-κB transcriptional activity. In support of this conclusion, PI3K/Akt/mTOR signaling has already been recognized to play a crucial role in inflammatory infiltration (60,61) and IR-induced cardiac injury (62,63). However, we cannot exclude the feasibility that aloperine regulates additional pathways other than the PI3K/Akt/mTOR signaling in the setting of IR-induced acute renal injury. In addition, the detailed mechanisms underlying aloperine repression of PI3K signaling remain to be elucidated and would be a major focus for future studies. It is worth noting that aloperine did not regulate Jak2/Stat3 signaling, a critical pathway for IL-6 and TGF-β transcription, and, therefore, IL-6 and TGF-β expression were not affected following aloperine treatment. However, previous studies also suggested that the transcription of IL-6 can be regulated by NF-κB as well (64). The confliction between our current report and previous studies could be caused by the differences in experimental system and cell types. Also, our data could be a consequence of interplay between NF-κB and Jak2/Stat3 signaling.
Programmed death of tubular cells is another critical characteristic feature relevant to IR-induced acute renal injury (65). Indeed, IR insult rendered tubular cells undergoing apoptosis along with upregulation of caspase-3. However, pretreatment of mice with aloperine prevented tubular cells from IR-induced apoptosis as evidenced by the significant reduction of TUNEL-positive tubular cells and decreased protein levels for caspase-3. This finding somehow is in conflict with studies in tumor cells in which aloperine was noted to promote cancer or tumor cell apoptosis (26,66). To confirm this observation, we cultured HK2 cells under hypoxic conditions in the presence of aloperine and similar results were obtained (data not shown). In the setting of cancer therapy, high doses of aloperine were employed to generate cytotoxicity for killing tumor cells, and therefore, it is likely that this discrepancy was caused by the differences of aloperine doses.
Given that oxidative stress caused by a burst of ROS generation and a decrease of antioxidants is a critical feature in IR-induced tubular apoptosis (44), we next examined the impact of aloperine on IR-induced ROS accumulation. As expected, IR insult induced a significant accumulation of ROS in the kidneys. However, pretreatment of mice with aloperine significantly prevented ROS accumulation as manifested by the reduction of fluorescence intensity. We then checked SOD activity, a critical enzyme responsible for the detoxification of ROS. It was noted that mice that had undergone IR insult manifested a significant reduction of SOD activity, while pretreatment of mice with aloperine almost completely prevented IR-induced suppression of SOD activity, suggesting that IR-induced ROS accumulation is at least in part caused by the impaired capability for ROS detoxification. Nevertheless, lipid peroxidation is unlikely involved in the induction of oxidative stress in our model as no difference was noted for MDA between all groups of mice. Of important note, in contrast to our observations in IR-induced acute renal injury, a previous study suggested that aloperine was able to reduce the content of MDA in hepatic tissue of intoxicated animals without affecting SOD activity (28). This discrepancy might be due to the differences of animal models employed.
To further address the mechanisms relevant to enhanced SOD activity and repressed tubular apoptosis, we checked AP-1 activity as it is a crucial factor responsible for SOD expression and antiapoptotic Bcl-2 transcription (45,67). Excitingly, renal lysates originated from aloperine-pretreated mice displayed a significant increase for nuclear AP-1 subunit c-Fos along with enhanced Bcl-2 expression. To confirm those results, we further examined HK2 lysates that had undergone hypoxic insult. Indeed, the addition of aloperine significantly enhanced hypoxia-induced nuclear c-Fos and antiapoptotic Bcl-2 expression, which was completely abrogated by the addition of TanIIA, an AP-1 inhibitor. Collectively, our data support that aloperine regulates AP-1 activity, by which it enhances SOD expression to promote ROS detoxification along with an increase of antiapoptotic Bcl-2 expression, thereby protecting tubular cells from IR-induced apoptosis. Interestingly, blockade of AP-1 activity in RAW264.7 cells by the addition of TanIIA almost completely abolished aloperine-induced IL-10 expression, indicating that aloperine promotes IL-10 expression by enhancing AP-1 activity as well. Of important note, PI3K/Akt signaling has been suggested to serve as a survival signal during renal IR (68) and, as a result, aloperine protection of IR-induced tubular apoptosis could be a compromised effect from the homeostasis of multiple signaling pathways.
In summary, we demonstrated convincing evidence that pretreatment of mice with aloperine provides protection against IR-induced acute renal injury as manifested by the attenuated inflammatory infiltration, reduced tubular apoptosis along with well preserved renal function. Mechanistic studies revealed that aloperine selectively represses IL-1β and IFN-γ expression by regulating PI3K/Akt/mTOR signaling and NF-κB transcriptional activity. Aloperine also regulates AP-1 activity, through which it not only enhances SOD and antiapoptotic Bcl-2 expression to prevent tubular cells from IR-induced apoptosis, but also promotes IL-10 expression to limit inflammatory response. Together, our data support that the administration of aloperine prior to IR insults such as renal transplantation could be a viable approach to prevent IR-induced injuries in clinical settings.
The authors declare that they have no competing interests as defined by Molecular Medicine, or other interests that might be perceived to influence the results and discussion reported in this paper.
This work was supported by grants from the National Natural Science Foundation of China (81130014, 81471046 and 81530024), the European Foundation for the Study of Diabetes (EFSD)/Chinese Diabetes Society (CDC)/Lilly Program for Collaborative Diabetes Research between China and Europe, and Innovative Funding for Translational Research from Tongji Hospital.
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