Propofol Stimulates Immune Activity and Decreases Inflammatory Cytokines via NF-κB-Mediated JAK1-STAT3 Pathway in Gastric Cancer Patients Undergoing Radical Surgery


 BackgroundPropofol is the most commonly used general anesthesia for patients with gastric cancer undergoing radical surgery. Studies have suggested that propofol exerts beneficial effects on the immune function of patients with cancer. However, the potential mechanism underlying propofol-mediated immune regulation remains to be elucidated. The present study investigated the regulatory effects of propofol on immune function in patients with gastric cancer undergoing radical surgery. MethodsELISA, reverse transcription-quantitative polymerase chain reaction, western blotting, gene transfection and immunohistochemistry were used to analyze the effects of propofol on gastric cancer cells.ResultsResults demonstrated that propofol general anesthesia resulted in an increased percentage of cluster of differentiation (CD)4+ and CD8+ cells, increased serum concentrations of interleukin (IL)-2 and tumor necrosis factor (TNF)-α, decreased serum concentrations of IL-1β and IL-8 following propofol general anesthesia in gastric cancer patients undergoing radical surgery compared with midazolam. In addition, propofol general anesthesia induced an imbalance in T helper (Th)1/Th2 cells, increased the number of natural killer cells and B cells, decreased the expression of prognostic factors, and improved tumor metastasis, recurrence and survival in patients with gastric cancer compared with midazolam. Furthermore, immunohistochemistry demonstrated that propofol downregulated nuclear factor (NF)-κBp65, and upregulated Janus kinase 1 (JAK1) and signal transducer and activator of transcription 3 (STAT3) expression level in gastric cancer tissues, downregulated the protein expression levels of NF-κBp65, JAK1, STAT3, TNF-α, IL-1β and IL-8 protein expression in gastric cancer cells isolated from gastric cancer tissues. NF-κBp65 overexpression inhibited propofol-mediated upregulation of JAK1, STAT3, TNF-α, IL-1β and IL-8 expression in gastric cancer cells. ConclusionsThese data indicate that that propofol may increase the number of T cells, stimulate T-cell proliferation, upregulate IL-2 and TNF-α expression, and enhance immune function via the NF-κB-mediated JAK1-STAT3 signaling pathway in patients with gastric cancer undergoing radical surgery.


Conclusions
These data indicate that that propofol may increase the number of T cells, stimulate T-cell proliferation, upregulate IL-2 and TNF-α expression, and enhance immune function via the NF-κB-mediated JAK1-STAT3 signaling pathway in patients with gastric cancer undergoing radical surgery.

Background
Gastric cancer is the one of the most common causes of cancer-associated mortality (1,2). Gastric carcinogenesis is thought to be associated with genetic factors, as well as numerous environmental factors (3,4). Gastric cancer is also associated with higher morbidity and mortality rates compared with other types of digestive system-derived carcinoma (5,6). Gastric signet ring cell carcinoma frequently presented with diffuse and in ltrating myositis, which increases the di culty of clinical diagnosis and treatment for patients with suspected gastric cancer (7). At present, apoptosis resistance of gastric cancer was inevitable in the development of cancer progression, thus increasing the risk of metastasis in patients with gastric cancer. Previous studies indicated that surgery combined with immunotherapy was more e cient for the treatment of gastric cancer (8,9); therefore, stimulating the immune function of patients with gastric cancer may be bene cial for tumor eradication.
Currently, propofol is one of the most widely used general anesthetics. Propofol attenuated the surgical stress-induced adverse immune response, which has short-term consequences in clinical patients receiving cancer or cardiac surgery (10,11). A previous study revealed that propofol attenuated the surgical stress-induced adverse immune response better than iso urane anesthesia, and can regulate the T helper cell (Th)1/Th2 ratio following surgery in patients undergoing craniotomy (12). A retrospective analysis demonstrated that general anesthesia with propofol was associated with a higher overall 1-year survival rate than sevo urane in patients following radical colon and breast cancer surgery (13). A randomized trial also demonstrated that patients receiving propofol exhibited increased interleukin (IL)-2/IL-4 and cluster of differentiation (CD)4 + /CD8 + T cell ratio compared with in des urane-treated patients undergoing breast cancer surgery (14). Interleukin-1 alpha (IL-1α) plays an important role in tumorigenesis and angiogenesis of gastric cancer and the interleukin-1 receptor antagonist (IL-1RA) signi cantly inhibited the proliferation and migration of human gastric cancer cells (15). Data found that suppression of IL-8-Src signaling inhibited adhesion, migration and invasion of gastric cancer cells (16).
Nevertheless, to the best of our knowledge, no study has evaluated the effects of propofol on perioperative immune function in patients undergoing radical surgery for gastric cancer.
The present study aimed to investigate the effects of propofol anesthesia on immune function, including the expression of cytokines [IL-1β, IL-8, IL-2 and tumor necrosis factor (TNF)-α], the number of immune cells (CD4 + and CD8 + cells), and the balance between CD4 + /CD8 + and Th1/Th2 in patients undergoing radical surgery for gastric cancer. In addition, the immunoregulatory effects of propofol were compared pre-and post-operation in patients with gastric cancer. The ndings provide evidence to suggest that propofol may increase the production of immune cells for patients undergoing radical surgery for gastric cancer.

Methods
Ethics statement.
The present study (registration number: TPHRZ20150412) was approved by the Ethics Committee of the Qiqihar Medical University. All patients agreed to the use of their samples in scienti c research and provided written informed consent.
Patients. Between May 2015 and July 2016, patients (n = 122) with gastric cancer requiring radical surgery provided written informed consent prior to the trial. The mean age of clinical patients was 48.5 ± 10.5 years, and the mean body weight was 70.4 ± 10.6 kg. The number of male and female patients (n = 61/61) was almost equal. The operation time for each patient was ~ 3 h. All of the patients were scheduled for radical surgery under propofol general anesthesia (3 mg/kg/h, n = 61) or midazolam anesthesia (3 mg/kg/h, n = 61). For all patients, there was no history of endocrine, immune or circulatory system diseases. Patients with gastric cancer with major surgical complications were excluded from the present study. The standardized protocols for the surgical procedure and postoperative patient care were used to minimize any difference in surgical impact among patients.
Patient survival, tumor metastasis and recurrence. A total of 22 patients with gastric cancer received propofol general anesthesia (3 mg/kg/h) prior to surgery, and were followed-up for 60 months. In addition, another 22 patients with gastric cancer (men/women, 10/12; mean age, 36.5 years; mean body weight, 57.5 kg) received midazolam general anesthesia. Patient survival, and tumor metastasis and recurrence were recorded within the 60-month follow-up period. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis. Total RNA was extracted from total peripheral blood samples using TRIzol® reagent (Invitrogen; Thermo Fisher Scienti c, Inc., Waltham, MA, USA), and RNA purity and quantity were detected by ultraviolet spectrometry. PCR speci c primers (Table I) were synthesized by Invitrogen; Thermo Fisher Scienti c, Inc. cDNA was synthesized by RT using RNeasy Mini kit (Qiagen Sciences, Inc., Gaithersburg, MD, USA), according to the manufacturer's protocol, after which qPCR was conducted using a FastStart Universal SYBR Green Master (cat. no. 4913850001; Roche Diagnostics, Basel, Switzerland) and a LightCycler 480 Real-Time PCR system (Roche Diagnostics) under the following conditions: 95˚C for 30 sec, followed by 28 cycles at 95˚C for 30 sec, 56˚C for 45 sec and 72˚C for 35 sec, and a nal extension step at 75˚C for 10 min. Quanti cation cycle (Cq) values of standard samples were calculated based on the internal reference gene β-actin. PCR results were quantitatively analyzed using the 2 −∆∆Cq method, as described previously (17). The Th1/Th2 balance was analyzed using the IL-2 + TNF/IL-4 + IL-10 ratio.
Blood cell counts. A complete blood count was performed on all blood samples using an automated hemoanalyzer (cat. no. WD-5000; Weier Medical Equipment Co., Ltd., Changchun, China), according to the manufacturer's protocol. Total and differential leukocyte, lymphocyte, monocyte and neutrophil counts were determined as described previously (18).
Western blotting. Gastric cells and NF-κB-overexpressed cells were lysed in radioimmunoprecipitation assay buffer (M-PER reagent for cells; Thermo Fisher Scienti c, Inc.) and were homogenized at 4˚C for 10 min. Protein concentration was measured using a bicinchoninic acid protein assay kit (Thermo Fisher Scienti c, Inc.). Subsequently, protein extracts (20 µg) were separated by 12.5% SDS-PAGE and were then transferred to polyvinylidene uoride membranes (EMD Millipore, Billerica, MA, USA). The membranes were incubated in blocking buffer (5% milk) for 2 h at 37˚C prior to incubation with primary antibodies at 4˚C overnight. The primary rabbit anti-human antibodies used in the immunoblotting assays were: NF- Abcam) and β-actin (1:2,000, cat. no. ab8226; Abcam). After incubation, the membranes were washed three times in Tris-buffered saline containing 1.0% Tween (TBST) and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) monoclonal antibody (1:5,000, cat. no. PV-6001; OriGene Technologies, Inc., Beijing, China) for 1 h at 37˚C. After three washes with TBST, the membranes were developed using a chemiluminescence assay system (Roche Diagnostics) and exposed to Kodak lms (Kodak, Rochester, NY, USA). Densitometric semi-quanti cation of the immunoblots was performed using Quantity-One software (version 1.2; Bio-Rad Laboratories, Inc., Hercules, CA, USA).
Immunohistochemistry. Immunohistochemical analysis was performed as described previously (21). Gastric cancer tissues were obtained from patients and were xed with 10% formalin for 2 h at 25˚C. Subsequently, the tissues were washed with PBS for 10 min, incubated with xylene for 5 min and incubated with an ethanol gradient (80, 90, 95 and 100%) for 3 min at 25˚C. Tissues were then embedded in para n at 56˚C and para n-embedded gastric cancer tissues sections (4 µm) were prepared for further analysis. The para n-embedded sections were treated with hydrogen peroxide (3%) for 10-15 min at 37˚C and were hydrated in a decreasing series of ethanol. Antigen retrieval was performed using an antigen retrieval kit (cat. no. ab93684; Abcam) at 65˚C for 10 min. Subsequently, 5% bovine serum albumin (Sigma-Aldrich; Merck KGaA) was used to block nonspeci c binding at 37˚C for 2 h and tissue sections were incubated with the following primary antibodies at 4˚C for 12 h: NF-κBp65 (1:1,200, cat. no. ab16502; Abcam), JAK1 (1:1,200, cat. no. ab47435; Abcam) and STAT3 (1:500, cat. no. ab68153; Abcam). The tissue sections were then washed three times and incubated with a HRP-conjugated IgG monoclonal antibody (1:2,000, cat. no. PV-6001; OriGene Technologies, Inc.) for 12 h at 4˚C. Sections were incubated with diaminobenzidine (Sigma-Aldrich; Merck KGaA) at room temperature for 10 sec, in order to detect positive signals, and six random views were observed under a microscope. Images were recorded using an inverted light microscope (Olympus Corporation, Tokyo, Japan). Protein density was analyzed using ImageJ software 4.6 (National Institutes of Health, Bethesda, MD, USA).
Statistical analysis. All data are presented as the means ± standard deviation of triplicate experiments, and were analyzed by SPSS 19.0 statistical software (SPSS, Inc., Chicago, IL, USA). The comparisons of means between two matched groups were conducted using a paired Student's t-test. Unpaired t-test or ANOVA were used to compare statistical difference between two groups. One-way ANOVA analysis followed by Tukey's test was used to compare statistical difference among multiple groups. P < 0.05 was considered to indicate a statistically signi cant difference.

Results
Clinicopathological features of patients with gastric cancer. In the present study, the propofol group comprised 61 patients with gastric cancer, and midazolam comprised 61 patients with gastric cancer. There were no signi cant differences in heart rate, respiration rate and body temperature between propofol and midazolam group (Table II). Data revealed that post-operation of propofol had less pain that midazolam for patients with gastric cancer.
Effects of propofol on immune cells. The number of leukocytes in patients with gastric cancer was increased 4 days after propofol general anesthesia compared with pre-operation and midazolam (Fig. 1A). In addition, the number of lymphocytes was decreased 4 days after propofol general anesthesia compared with midazolam (Fig. 1B). The number of monocytes was increased 4 days after general anesthesia compared with midazolam (Fig. 1C). Neutrophil levels were also increased 4 days after propofol general anesthesia compared with midazolam (Fig. 1D). These ndings indicated that propofol general anesthesia regulated immune cells in patients with gastric cancer undergoing radical surgery.
Effects of propofol on cytokine expression. The effects of propofol on the expression levels of cytokines were analyzed 4 days after propofol general anesthesia in patients with gastric cancer undergoing radical surgery. Serum levels of IL-2 and TNF-α were increased following propofol general anesthesia compared with midazolam (P < 0.01; Fig. 2A and B). Compared with pre-operation, the serum concentrations of IL-1β and IL-8 were decreased in patients following propofol treatment compared with midazolam (P < 0.01; Fig. 2C and D). These ndings suggested that propofol general anesthesia regulated cytokine expression in patients with gastric cancer undergoing radical surgery.
Effects of propofol on CD4 + , CD8 + and CD4 + /CD8 + cells. As shown in Fig. 3A and B, there was a statistically signi cant increase in CD4 + and CD8 + T cells following propofol anesthesia compared with midazolam (P < 0.05). In addition, the CD4 + /CD8 + T cell ratio was increased 4 days after propofol anesthesia compared with midazolam (P < 0.05; Fig. 3C). These ndings suggested that propofol general anesthesia increased the percentage of CD4 + and CD8 + cells in patients with gastric cancer undergoing radical surgery.
Effects of propofol on the Th1/Th2 balance. ELISA was used to determine the effects of propofol anesthesia on the serum levels of Th1 and Th2 cytokines. The results indicated that the mRNA expression levels of Th1 cytokines, IL-2 and TNF-α, were increased 4 days after propofol anesthesia (P < 0.01; Fig. 4A). In addition, the expression levels of Th2 cytokines, IL-4 and IL-10, were increased 4 days after propofol anesthesia in patients with gastric cancer undergoing radical surgery (P < 0.05; Fig. 4B).
However, the Th1/Th2 ratio was decreased 4 days after propofol anesthesia (P < 0.05; Fig. 4C). These ndings suggested that propofol general anesthesia regulated cytokine expression in patients with gastric cancer following radical operation.
Effects of propofol on prognostic factors. The prognostic factors Cav-1, PDL-1, MECP2 and YB-1 were evaluated in patients with gastric cancer undergoing radical surgery. The serum levels of Cav-1, PDL-1, MECP2 and YB-1 were decreased in patients with gastric cancer following propofol anesthesia compared with midazolam (P < 0.01; Fig. 5A-D). These ndings indicated that propofol general anesthesia may decrease prognostic factors in patients with gastric cancer undergoing radical surgery.
Effects of propofol on tumor metastasis, recurrence and survival. The effects of propofol general anesthesia on tumor metastasis, recurrence and patient survival were analyzed in the present study. As shown in Fig. 6A, propofol general anesthesia inhibited gastric cancer metastasis following radical surgery compared with midazolam. In addition, the ndings indicated that gastric cancer recurrence was decreased following propofol general anesthesia in patients undergoing radical surgery (Fig. 6B). Notably, survival was prolonged by propofol general anesthesia in patients with gastric cancer undergoing radical surgery compared with midazolam (Fig. 6C). These results suggested that propofol general anesthesia improved tumor metastasis, recurrence and survival for patients with gastric cancer undergoing radical surgery during a 60-month follow-up period.
Effects of propofol on the NF-κB-mediated JAK1-STAT3 signaling pathway. A previous study demonstrated that NF-κB can regulate responses in the immune system (22). Therefore, the present study further analyzed the potential mechanism underlying the effects of propofol on gastric cancer cells. The results revealed that propofol downregulated NF-κBp65 expression, and upregulated JAK1 and STAT3 expression in gastric cancer tissues (Fig. 7A). Western blotting demonstrated that propofol treatment decreased the protein expression levels of TNF-α, IL-1β and IL-8 in gastric cancer cells (Fig. 7B).
Furthermore, propofol downregulated NF-κBp65, and upregulated JAK1 and STAT3 protein expression in gastric cancer cells (Fig. 7C). The present study also demonstrated that NF-κBp65 overexpression canceled propofol-induced upregulation of JAK1 and STAT3 protein expression in gastric cancer cells compared to cells transfected empty vector (Fig. 7D). In addition, propofol-induced decreases in TNF-α, IL-1β and IL-8 expression were attenuated by NF-κBp65 overexpression in gastric cancer cells (Fig. 7E).
These results indicated that propofol regulated immune responses via the NF-κBp65-mediated JAK1-STAT3 signaling pathway.

Discussion
Propofol has been reported to present fewer adverse and toxic side-effects, and exhibits reduced toxicity in the heart and central nervous system compared with midazolam (23). In addition, it has been demonstrated that propofol general anesthesia increased the percentage of CD4 + and CD8 + cells in patients undergoing laparoscopic radical hysterectomy for cervical cancer (24). Therefore, it was hypothesized that propofol increased the production of cytokines and provided protection for circulating lymphocytes during the perioperative period for patients undergoing radical surgery for gastric cancer. Although a previous report indicated that propofol anesthesia had less of an effect on immune function in patients with lung adenocarcinoma (25), the present study demonstrated that propofol general anesthesia was able to regulate immune cells and cytokine production in patients with gastric cancer undergoing radical surgery.
Gastric cancer is an important health problem, which is particularly intractable due to the existence of gastric tumor cells in the hydrochloric acid-containing gastric juice, which represents a relatively more extreme acidic environment compared with other human tissues (26,27). In recent years, the inhibitory effects of propofol on human cancer cells have been widely studied. It has been revealed that propofol suppressed invasion of human lung cancer cells by downregulating aquaporin-3 and matrix metalloproteinase-9 expression (28). Notably, propofol anesthesia attenuated the surgical stress-induced adverse immune response, which may regulate the Th1/Th2 ratio (12). The present study indicated that propofol anesthesia not only had an anesthetic effect, but also stimulated immune regulation in patients undergoing radical surgery for gastric cancer. Zhou et al demonstrated that propofol was able to attenuate sevo urane-induced cellular injury of human peripheral lymphocytes (29). The present ndings indicated that propofol general anesthesia decreased the levels of lymphocytes, and increased monocytes and neutrophils after general anesthesia compared with pre-operation for patients with gastric cancer undergoing radical surgery.
Several kinds of cancer surgeries with propofol-based total intravenous anesthesia may be associated with improved survival in gastric cancer patients who undergo resection (30). Propofol general anesthesia achieved a more stable hemodynamics and a shortened time to awakening (30). Data in the current study found that propofol general anesthesia shortened the hospital stays and time to awakening compared to previous study. Propofol sedation presented more bene ts during endoscopic treatment for early gastric cancer compared to midazolam (31). Notably, our study found an association between propofol anesthesia and an increased anti-in ammatory cytokines. This may be due to the fact that propofol anesthesia induced activated immune functions. Propofol can promote the secretion of insulin during radical gastrectomy, and inhibited the excessive secretion of cortisol and hyperglycemia (32). In this study, propofol decreased serum levels of Cav-1, PDL-1, MECP2 and YB-1 for gastric cancer patients undergoing for gastric surgery. However, the in uence of propofol on stress responses did not investigate in patients undergoing for gastric surgery.
IL-2 and IL-4 were associated with Th1 and Th2 cells, and the balance between Th1 and Th2 cells serves a signi cant role in patients with cancer (33). A previous study revealed that propofol anesthesia regulated the Th1/Th2 balance in spinal cord injury (34). The effects of propofol and dexmedetomidine anesthesia have also been detected on the Th1/Th2 balance in rat spinal cord injury (34). The present study demonstrated that the Th1/Th2 ratio was decreased after propofol anesthesia in patients with gastric cancer undergoing radical surgery, which may contribute to a reduction in in ammation. In addition, a previous study reported that propofol anesthesia for breast cancer surgery induced a favorable immune response in terms of preservation of IL-2/IL-4 and CD4 + /CD8 + T cell ratio during the perioperative period (14). The present study revealed that IL-2 and TNF-α expression were signi cantly increased following propofol anesthesia. These ndings suggested that propofol anesthesia may increase the percentage of CD4 + and CD8 + cells in patients undergoing radical surgery.
A previous study demonstrated that continuous infusion of midazolam affected immune function by decreasing the expression levels of IL-1β, IL-8 and TNF-α in pediatric patients post-surgery (35). Romano et al revealed that IL-2 immunotherapy enhanced tumor-in ltrating lymphocytes in patients with gastric cancer (36). The present study demonstrated that propofol anesthesia decreased the serum levels of IL -1β and IL-8 in patients undergoing radical surgery. Furthermore, A. Ní Eochagáin et al showed that neutrophil-lymphocyte ratio increased signi cantly in the propofol-paravertebral breast cancer patients (37). Results in this study observed the increasing neutrophil serum level in propofol group. However, neutrophils did not incarcerated on postoperative day 4 in the midazolam group. De nitely, neutrophil stimulated by propofol may contribute to the chemotactic attraction of suppressive gastric cancer cells. A previous study found that there could be a much de ned axis where IL-8 plays an important role in the recruitment of certain lymphocyte populations and tumor development, including the way in which tumors are capable of developing metastasis (38). In this study, although we observed the increasing of neutrophil levels after propofol general anesthesia, the serum concentration of IL-8 was decreased in patients following propofol treatment. These mechanisms mediated by IL-8 may be relevant in the establishment of immune system that helps the immune cells to monitor the tumor microenvironment. Although TNF-α and IL-2 were proin ammatory cytokines for patients with gastric cancer, IL-2 stimulated the host reaction against tumor tissues by lymphocyte/eosinophil in ltration (39), and TNF-αinduced apoptosis of human gastric cancer cells via upstream caspase-3 protease activation (40). The present study reported that propofol anesthesia increased the serum levels of IL-2 and TNF-α in patients undergoing radical surgery, which may enhance anticancer therapy for patients with gastric cancer. However, the in vitro assay demonstrated that propofol decreased TNF-α in gastric cancer cells. These differences may be due to the differences between intracellular in ammation and systematic in ammatory responses. These outcomes indicated that propofol anesthesia exerted bene cial effects for patients with gastric cancer undergoing radical surgery. However, the sample size of this clinical study was small and needed further clarify in our future work.
Cav-1 promoted bladder cancer metastasis, and is therefore considered a potential therapeutic target in invasive bladder cancer (41). In addition, it has been reported that tumor expression of PDL-1 was associated with poor prognosis in patients with gastric cancer (42). This study reported that the serum levels of Cav-1 and PDL-1 were decreased in patients with gastric cancer following propofol anesthesia. Furthermore, dysregulated expression of MECP2 was correlated with clinicopathological parameters in the development of gastric cancer (43). A previous study also indicated that silencing the YB-1 gene led to inhibition of cell migration in gastric cancer in vitro (44). In the present study, propofol anesthesia decreased MECP2 and YB-1 serum levels in patients with gastric cancer, thus suggesting that propofol may be a potential anesthetic with anti-metastatic activity for patients with gastric cancer. A previous study revealed that inhibition of NF-κB is required for properly balanced immune responses, and it appeared to be evolutionarily conserved (45). In addition, it has been reported that propofol inhibited invasion and growth of ovarian cancer cells by regulating the NF-κB signaling pathway (46). In the present study, propofol was shown to downregulate NF-κB expression in gastric cancer tissues and cells. Furthermore, Lu et al demonstrated that the JAK1/STAT3 pathway was involved in the protective effects of propofol against hypoxia-induced in ammation and apoptosis in BV2 microglia (47). Chen et al found that activation of JAK1/STAT3 signaling inhibited tumorigenesis and induced cell apoptosis repression in gastric cancer (48). Notably, inhibiting the activation of the JAK1/Stat3 pathway is a practical anti-tumor approach to restrain tumor progression in in gastric cancer (49). The present study also reported that propofol regulated immune function via the NF-κB-mediated JAK1/STAT3 signaling pathway in gastric cancer cells. These results provided a potential target for the regulation of immune function in patients with gastric cancer undergoing radical surgery.

Conclusion
In conclusion, treatment with propofol during radical surgery exerted favorable effects on the immune system, particularly with regards to the Th1/Th2 balance and CD4 + /CD8 + T cell ratio, during the perioperative period. The present ndings indicated that propofol anesthesia decreased the Th1/Th2 ratio, which attenuated adverse effects on the immune system during radical surgery for patients with gastric cancer. Further studies are required to determine the clinical implications of using propofol compared with other types of general anesthesia, such as etomidate, in a large population.

Declarations
The authors declare that they have no con ict of interests.