Effects of NF-?B and the role of inflammatory response factors on the hepatocytic injury after Trans-arterial embolization – An experimental studyBimbadhar Valluru, MD†1, Wang Bei-Ran, MS†1, Du Wei, MD, Ph.D*1, Yu Yi-Jun, MS1,Wu Chun-Hua, MS1, Kalyan Sharma, MD11 Bimbadhar Valluru, MD, Department of Radiology, the First Affiliated Hospital of Dali University. E-mail: [email protected] Bei-ran, MS, Department of Radiology, the First Affiliated Hospital of Dali University.
E-mail: [email protected] Wei, MD, Ph.D., Department of Radiology, the First Affiliated Hospital of Dali University.E-mail: [email protected] Yi-Jun, MS, Department of Radiology, the First Affiliated Hospital of Dali University.
E-mail: [email protected] Chun-Hua, MS, Department of Radiology, the First Affiliated Hospital of Dali University. E-mail: [email protected] Sharma, MD, Department of Radiology, the First Affiliated Hospital of Dali University.
E-mail: [email protected] †These authors contributed equally to this workFirst Author- Bimbadhar ValluruCo-First Author- Wang Bei-ranSecond Author(s)- Yu Yi-Jun, Wu Chun-Hua, Kalyan Sharma*Corresponding author: Dr. Du Wei, Department of Radiology, the First Affiliated Hospital of Dali University. Dali 671000 Ph: +86-18608855588. Keywords: Trans arterial emobolization; Hepatocellular carcinoma; NF-?B; TNF-?; IL-10; IR; TACE. AbstractObjective: To investigate the effects of NF-?B and the role of inflammatory response factors on the Normal liver tissue around the tumors in rabbit VX2 hepatocellular carcinoma models after TAE. Methods: VX2 liver cancer was implanted into 70 healthy New Zealand rabbits; the Computed tomography (CT) and Magnetic resonance imaging (MRI) examination were performed on the 21 days after modeling.
A total of 60 VX2 hepatoma models were randomly chosen and divided into three groups, 20 in each group: TAE GROUP, CONTRAST/ANGIOGRAPHY GROUP, and CONTROL GROUP respectively. TAE group was treated with Trans-arterial embolization (TAE) and the Contrast group was subjected to hepatic arteriography and DSA, while the Control group served as a control group. The immune-histochemistry analysis was used to detect the expression of NF-?B in normal liver tissue around the tumors of each group. The levels of inflammatory factors like TNF-?, IL-10 of each group were detected by Enzyme-linked immunosorbent assay (ELISA) in the normal liver tissue around the tumor. SPSS 22.0 statistical analysis software package for statistical analysis, measurement data using t-test, count data were compared using the ?2-test; P <0.
05 was considered as statistically significant. Results: The successfully established models were 60 (60/69), and the success rate was 85.7%. The positive expression rates of NF-?B in the TAE group, contrast group, and control group were 75% (15/20), 35% (7/20) and 20% (4/20) respectively.
The concentrations of TNF-? were 11.72 ± 0.65 (ng / L), 9.
60 ± 0.31 (ng / L) and 8.82 ± 0.81 (ng / L) respectively.
The concentrations of IL-10 were 2.18 ± 0.13 (ng / L) 1.67 ± 0.12 (ng / L), 1.66 ± 0.10 (ng / L).
Compared with the control group and contrast groups, TAE group showed a significant increase in the expression of NF-?B, and the levels of TNF-? and IL-10 were also increased. Conclusion: The injury of liver function after TAE may be due to the interaction of NF-?B and inflammatory factors-TNF-? and IL-10.AbbreviationsTAE-Trans-arterial embolization; CT- Computed Tomography; MRI-Magnetic Resonance Imaging; DSA- Digital Substraction Angiography; ELISA- Enzyme Linked immunsorbent assay; NF-?B- Nuclear Factor kappa B; TNF-?-Tumor necrosis factor alpha; IL-10- Interleukin-10.
IntroductionHepatocellular carcinoma (HCC) is the leading cause of cancer-related deaths in the world. While metastases are more common in westren countries, primary liver cancers are frequently diagnosed in Asia and Africa. The incidence and mortality rate of liver cancer patients in China, also account for 50% of the world 1. Radical segmental resection or hepatectomy is associated with a better prognosis for patients with primary liver cancer. It is the preliminary choice of management, but only 15-20% (considerably lower resection rates) of the patients are suitable for surgical resection because of insidious onset, rapid growing tumors and early metastasis1, 2; while most patients lose the opportunity because of the extent of tumor is beyond 40% of residual liver tissue remaining at the time of diagnostic assessment and poor hepatic function is the main factor responsible for higher morbidity and mortality 3. But local ablative treatments, such as MR-guided or laser interstitial thermotherapy (LITT), microwave coagulation or radiofrequency ablation, are also more frequently used techniques. However, only a few patients are candidates for these treatments 4,5,6. Transcatheter arterial embolization (TAE) is a minimally invasive, loco-regional interventional therapeutic procedure which is also referred to as liver-directed therapy 7.
It has been widely used in the treatment of intermediate and advanced HCC patients as it intervenes to focally target hepatocellular carcinoma within a liver. Though commonly used, it is found that patients often have varying degrees of hepatic dysfunction after undergoing TAE, and consequently has higher mortality rates owing to the eventual development of liver failure 8. The bigger problems to solve for interventional surgeons are to find techniques on how to reduce the extent of the liver injury or even avoid TAE induced hepatic dysfunction as they have become the key in improving the prognosis of patients. Previous studies showed that Nuclear factor- kappa B (NF- ?B), and inflammatory reaction (IR) plays an important role in the process of cell necrosis 9. Other inflammatory factors like Tumor Necrosis Factor-Alpha (TNF-?) and Interleukin-10 (IL-10) are also regulated by NF- kappa B (NF- ?B) 10.
The VX2 liver cancer model of liver cancer is commonly used to evaluate the efficacy of locoregional anticancer therapy experimentally 11. VX2 tumors can be induced by the Shope cottontail rabbit papillomavirus and is an anaplastic squamous cell carcinoma 12. These tumors grow rapidly and produce gycolytic large lesions that are appreciable on radiological imaging. So interventional radiologists have found this tumor to be a very uselful surrogate in the study of loco-reigonal liver directed therapies, mainly for Hepatocellular carcinoma as it has similarities to advanced human cancers 13. In this study, we set up rabbit VX2 hepatoma models in parallel to TAE therapy module and observed the expression of NF-?B in the paracancerous tissue as well as the changes in the content of TNF-? and IL-10 around the tumor. The main aim of this study is to explore this possible role of NF- ?B and inflammatory factors like TNF-? and IL-10 in stimulating hepatocytic injury after TAE.
Materials and MethodsMaterialsExperimental AnimalsTwo pre-modeled rabbits with subcutaneous VX2 tumors in the hind limbs were donated by Affiliated Hospital of Southeast University. 70 healthy New Zealand white rabbits, both male and female with an average weight of 2.8 +/-0.28 kgs (median-2.91 kgs) were provided by the Experimental Animal Center of Dali University; also were fed regularly ad libitum by the designated personnel. All the rabbits are in good condition, evaluated prior to experimentation/ modeling as per guidelines set by the Biomedical Ethical committee of Affiliated Hospital of Dali University.Essential Equipment, experimentation instruments, and drugsThe whole experiment utilized the following materials that were pre-equipped such as 16 row Spiral Computed Tomography Unit (Phillips), 3.0 Tesla Magnetic Resonance Imaging System (Toshiba Taitan), Digital subtraction Angiography (DSA) Unit, Interventional Surgery Unit (GE INNOVA-3100IQ ), all the units maintained at The First Affiliated Hospital of Dali University; predesigned experimental rabbit operation table and surgical drapes suitable for rabbits; consumables like multiple gauge syringes, ophthalmic scissors & forceps, kidney trays, plastic containers, Sterile kiosk (solitary workstation for tumor thawing and isolation), arterial clamps, 18G puncture needle, 4F cobra C3 hyrophilic angiographic catheter, 3F, 2.
4F sp microcatheter, 45- degree angled hydrophilic micro-guide seldinger wire; 3% phenobarbital sodium for anaesthesia, Gentamycin Sulfate Injection, 0.9% NaCl (Normal Saline) as required, iohexol for sterilization. NF-?B anti-mouse/rabbit polyclonal antibodies (purchased from MlBo Company, Wuhan), two anti-mouse/rabbit reagents, and DAB color reagent (purchased from Dako), IL-10, TNF-? anti-rabbit double sandwich test kit (purchased from Ancient Biological and Science Technological Laboratories Co.Ltd, Shanghai) were also pre-arranged.Methods(a) Preparation of the VX2 tumor models in rabbitsOne rabbit bearing a VX2 tumor on the hind legs was randomly selected for tumor extraction. 3% phenobarbital sodium (30mg/kg) was injected slowly (5ml/min) through the dorso lateral vein that runs along the pinna until sufficient anesthetic response was achieved. The rabbit was fixed in supine position on the operation table (predesigned); the surgical site was sterilized and draped, exposing the tumor on one of the hind limbs. A vertical incision was made to reach the epidermis and the tumor was carefully extracted isolating it from all the fascial attachments (Fig.
1 a). Surrounding necrotic tissue that spreads till subcutaneous layers were debrided surgically and the incision was closed by subcutaneous interrupted suture technique. The extracted VX2 tumor was placed into a kidney tray containing a mixture of 50 ml normal saline with 20000 IU of Gentamycin (Fig. 1 b). The tumor was then thawed at 36o to 37oC for 15 min using a thawing glass apparatus in the sterile kiosk; then transferred to a sterile plastic container with normal saline.
The resultant material was further cut into 1-mm3 pieces using ophthalmic scissors to prepare the tumor tissue suspension (Fig. 1 c). A consistent concentration of the mixture (15-20 tumor particles/ ml) from the tumor suspension was aspirated into a 1 ml syringe with an 18G puncture needle head. Six healthy rabbits were randomly selected (out of groups) and the tumor particles were injected subcutaneously (at 450 angle insertion) into the vastus medialis muscle of both the hind legs (0.5 ml for each side) for further preservation and inoculation. Experimental Animals- Preparation of the VX2 liver cancer modelsHealthy rabbits were randomly selected and anesthetized using the same method cited supra. The rabbits were sterilized and adequately draped exposing the abdomen.
To implant the VX2 carcinoma fragment into the liver, a standard sub-xiphoid laparotomy with an incision 3-5 cm in length was performed. The left lobe of the liver was pulled out gently by holding it with a sterile cotton gauge/ swab; a 1mm3 fragment of VX2 carcinoma was inoculated into the sub capsule of the lateral part of the left lobe of the liver because it has a larger surface area (Fig. 2 a). The puncture site of the liver was compressed with a sterile cotton gauze for 3-5 mins to prevent any unanticipated hemorrhage. Before the wound was closed in layers, 0.5 ml of Gentamycin (40000 IU) was dripped into the peritoneal cavity to prevent infection.
All the laparotomized VX2 liver cancer model rabbits underwent the same procedure and were injected with i.m gentamycin injection (40000 IU) 14,15 for consecutive 3 days, to prevent any post-surgical systemic infection. All the modelling was performed at the Experimental Animal Laboratory of Dali University.Grouping and procession of experimental modelsEach rabbit, modeled with VX2 liver cancer was scanned with CT & MRI after 7,14,21, 28 days of inoculation to track the tumor growth respectively. Total 60 rabbits were modeled and the success rate was 87% (60/69). The size, location, and radiographic charecteristics were recorded; the size of the lesion 1.5-2.0 cm on the 21st day, number of lesions, location and imaging characteristics consistent with primary liver cancer was considered as a standard for grouping to replicate different stages of HCC (Fig.
3 a-d). All the sixty rabbits were divided into 3 groups namely: TAE group, Angiography- contrast group, and Control group, accumulating 20 rabbits in each. For TAE group, a trans-arterial embolization (TAE) was performed by injecting 0.2 ml poppy seed iodized oil (Lipiodol) into the hepatic artery using “seldinger technique” at the interventional surgery unit (Fig. 4 a-b).
For the angiography-contrast group, angiography was performed through DSA by injecting non-ionic iodine contrast agent into the hepatic artery (Fig. 5 a-b). The control group did not receive any interventional procedures, but the progression of the tumor was monitored on CT/MRI for the consecutive 28 days. While working on conventional imaging (CT/MRI), DSA and interventional procedures, radiological protocols, post-processing techniques, and standard sterilization techniques were followed according to the guidelines set by The Department of Radiology, The First Affiliated Hospital of Dali University. Post procedural handling of Experimental Samples and RandomizationModeled rabbits from each group were euthanized after 28-35 days for further collection of samples. An incision was made laterally following along the left hypochondrium to mid-epigastrium and extending further along the midline till lower abdomen adequately to gain access to the site of the liver. The liver was completely isolated.
The tumor tissue along with surrounding residual normal liver tissue (paracancerous specimens) was separated and cut into pieces >2cm 16. Such para-cancerous tissues samples were collected from each group and marked accordingly. Gross pathological specimens for observation were collected (only 2 out of groups) which shows the successful implantation of the VX2 tumor and aprreciable hypoxic ischemic injury after TAE (Fig. 2 b-c).
Each sample was placed in a 5ml plastic test tube and transferred to a liquid nitrogen cooling tank for the next 15 minutes; then refrigerated at -800 C for the next 12 hours. Randomization was performed after specimen collections, and stored in a sealed, sequentially numbered containers and transferred for further analysis. HE Staining Thinly sliced sections from each sample were embedded with liquid paraffin and fixated with HE stain. Pathologic analysis of the liver provided a direct reference standard for comparing conventional imaging, angiography and TAE results among the groups to evaluate positivity rate, expression of NF-?B and inflammatory factors. The determination of expression of NF-?B All the samples were double stained and analyzed using Envision TM immuno-histochemical analysis technique to determine the expression of NF-?B.
In accordance to the positive expression of cells and positive color intensity at the detected protein expression sites as observed under 400X (10X10 grid) of magnification per FOV (field of view), scores were given as follows: Cell positive expression scoring: if the positive expression of cells was less than 5%- 0 points; if the cell expression of cells was less than 25%-1 point; 25-50%-2 points; and >50%-3 points were given respectively. Postive color intensity at the detected protein expression sites scoring: Points were given according to the color of the stain as intensity represents the amount of expressed protein. If the cells did not stain-o points; if cells stained with a light yellow-1 point; yellowish-brown-2 points; and dark brown-3 points were given respectively.
Finally, the average of the sum of a and b scores was taken as the basic criterion for determining the results. Overall average < 2 points is considered as negative expression (-); 2~3 points- a weak positive expression (+); 4~5 points- a moderately positive expression(++); and 6~7- a strongly positive expression (+++)&(++++) accordingly. The corresponding results are displayed in Fig. 6 a-f and (Table-1).
The detection of the protein content of TNF-? and IL-10Enzyme-linked immunosorbent assay (ELISA) was utilized to detect the level of TNF-? and IL-10 protein contents in all the specimens according to the instruction manual in the kit.Statistical analysisMeasurement data from all groups were analyzed using t-test, count data were analyzed using ?2- test and compared by Analysis of Variance (ANOVA) as moduled into SPSS Statistics (Ver.22.0; Chicago IL) software package for Windows (Microsoft). If P<0.05, the differences were statistically significant. ResultsAnalysis of NF-?B expression in VX2 liver cancer model groupsThe immune-histochemical analysis results showed that there was a strongly positive expression of NF-?B in 15 cases of the specimens from the paracancerous tissue of the TAE group, the positive rate was 75%; 7 cases had varied positive expression in the angiography group (35%) and the 4 cases were positive in the control group (20%). The Chi-square test (?2 – test) results as compared to each group showed that: 1) Angiography group as compared to the control group, (P>0.
05) there are no significant statistical differences; 2) TAE group as compared to the angiography group, (P<0.001) there were significant statistical differences and 3) TAE group as compared to the control Group Number of cases NF-?B expression Positivity Rate- + ++ +++ Control Group 20 16 1 2 1 20%Angiography Group 20 13 2 3 2 35%TAE Group 20 5 2 6 7 75%the group, (P=0.011), there were significant statistical differences as shown in (Table-1).Table-1 The expression of NF-?B in the paracancerous tissue specimensAnalysis of the detection of the protein content of TNF-? and IL-10The protein content of TNF-? and IL-10 were dominantly high in the TAE group than the angiography group, while the control group has the lowest expression.
The results of ANOVA are as follows: 1) Angiography group as compared to the control group,(P>0.05) there were no significant statistical differences; 2) TAE group as compared to the control group, (P<0.05), there were significant statistical differences; and 3) TAE group as compared to the angiography group, (P<0.05) there were significant statistical differences as shown in (Table-2). Group TNF-? IL-10Control Group 8.82+0.81 1.
66+0.10Angiography Group 9.60+0.
31* 1.67+0.12*TAE Group 11.72+0.65**# 2.18+0.13**#Table-2 The content of TNF-? and IL-10 in the cells adjacent to the cancerous tissue, *comparison between the contrast and the control group (P>0.
05), ** comparison of TAE group with the control group (P<0.05), # comparison of TAE group with the contrast group (P<0.05)Discussion The hepatic artery proper that arises from the common hepatic artery that runs alongside the portal vein and the common bile duct to form the portal triad provides the main vascular supply to the liver 17.
While normal liver tissue receives 75% of its blood supply from the portal vein and 25% from the hepatic artery, liver tumors are almost exclusively supplied by the hepatic artery 18,19. This different blood supply is used for the chemoembolization of liver tumors therapeutically. The liver parenchyma is rarely damaged by TACE because it obtains the majority of its blood supply from the portal vein, while blood supply to hepatic tumors originates predominantly from the hepatic artery 19.
Therefore, theoretically, embolization of the hepatic artery can lead to selective necrosis of the liver tumor leaving normal liver parenchyma virtually unaffected 20. But, TACE is based on the synergistic effect of arterial occlusion and local chemotherapy, which makes the tissue hypoxic and enhances the effectiveness of the chemotherapeutics 21,22. When the arterial blood flow is compromised due to blockage, in this scenario-embolization, the collateral blood flow from the portal vein compensates to maintain the normal function of the liver. About 99% of the liver tumors have feeding arteries that arise from the hepatic artery, and embolization of such branches provides a theoretical support of Trans-arterial embolization (TAE) in the treatment of hepatocellular carcinoma (HCC) 23. Therefore, theoretically, TAE provides another solution for patients with advanced liver cancers, most likely are inoperable at that stage as it improves the chances of survival by prolonging lifetime through assisted compensatory physiologic blood flow from the portal vein. However, practically TAE can have a drastical impact on the liver function even when embolization is done by super selective therapeutic interventions. Due to abundant vascular supply from the hepatic portal systems i.
e. hepatic sinusoid and the medial branch of the liver respectively, the intra hepatic uptake of embolic materail i.e inevitable entry of the lipodiol into the residual normal liver tissue that is adjacent to the cancerous tissue which gets deposited in the tiny arterioles and capillaries, finally causing low perfusion injury and triggers ischemic hypoxia within. This vascular disturbance stimulates the liver tissue to produce a large number of reactive oxygen species (ROS) and inflammatory factors namely TNF-? and IL-10 leading to a triggered series of complex endogenous reactions like apoptosis and necrosis, ultimately damaging liver function 24. So as a result, though cancer has been eliminated through embolization, such patients have a poor prognosis due to a severely compromised liver function which is considered to be the most serious and early complications after a TAE intervention. Recent studies have shown that TAE has some serious complications like an acute liver failure, liver infarction or hypoxic injury, post-embolisation syndrome etc, which is the main challenge of interventional surgeons and led to finding out how to prevent such complications 25.The inflammatory response (IR) is one of the first and most common pathophysiological reactions that can occur in any organ systems or tissues.
Early manifestations of an acute inflammation include rubor (redness), calor (heat), tumor (swelling), dalor (pain) and functio laesa (loss of function), which is presented as pyrexia (fastigium), leukocytosis and generalized symptoms. The factors of inflammation are mediated by TNF-? and the anti-inflammatory response is marked by IL-10 which could be mentioned as disproportionate resistance that occurs during an inflammatory process 26,27. In this scenario, these can be correlated with the raised levels of NF-?B, TNF-?, and IL-10 respectively, as a result of various stimuli such as TAE intervention, Angiography, and tumor implantation etc; while TAE group presented with the highest levels. This shows that TAE has indeed induced such inflammatory reaction.TNF-? is a cell signaling cytokine protein involved in systemic inflammation and is one of the cytokines that make up the acute phase reaction. It is produced chiefly by activated mononuclear macrophages 28. TNF being an endogenous pyrogen, it is able to induce fever, apoptotic cell death, cachexia, inflammation and to inhibit tumorigenesis.
The tumor necrosis factor receptor superfamily 1A (TNFR1A) is a ubiquitous membrane receptor that binds Tumor necrosis factor alpha (TNF-?) that triggers the destruction of lysosomes so that the endogenous cells are exposed to destructive enzymes 29. This receptor can also activate the transcription of NF-?B that mediates apoptosis and function as a regulator of inflammation. On the other hand, TNF- ? activates the tissue epithelial cells, increases the permeability of capillaries that lead to aggregation of edematous fluid which eventually hinders cell perfusion and gaseous exchange, as a result aggravating the tissue damage 30.
In our study, NF-?B was also elevated significantly, by which we can infer that NF-?B also triggered an inflammatory response following a different pathway. The strong toxic apoptotic effects of TNF-? are represented by the significant rise in their levels, we can infer that both NF-?B and TNF- ? may play an important role in inducing liver injury and consequently deteriorating the liver function after TAE.Interleukin 10 (IL-10), also known as human cytokine synthesis inhibitory factor (CSIF) that is primarily produced by monocytes (triggering PD-1) and type 2 T helper cells which have multiple, pleiotropic effects in immunoregulation and inflammation. It can produce reliable immunosuppressive effects on endothelial cells and that has shown to inhibit tumor metastasis 31,32, and strong anti-inflammatory effects on macrophages that eventually reduce tissue damage 33. When the body has an physiologic stress that occurs due to a pathology (stimulus) from various etiologies, the content of the inflammatory factors like TNF-? will increase, that ultimately causes cell degeneration and destroy the target local tissue cells. On the other hand, the potential of one’s body ability to ward off a disease (resistance) is mediated by anti-inflammatory factors like IL-10 which redirects to remove all the involved inflammatory factors, thereby reducing the influential damage, in an anticipation to restore the cell physiology to normal 33. So theoretically, if pro-inflammatory and anti-inflammatory factors are at equilibrium, the internal environment in the target cell will relatively be stable and such injury can be repaired eventually; on the contrary, if any one of them is overexpressed as dependant on conditions like the tumor size, stage of cancer (early-mid-late), metastatic spread, peritonitis, ascites, blood flow rate/pressure-the equilibrium cannot be attained, and finally aggravating the damage of target cells 34. In our study, we found that TAE group has a large number of neutrophils infiltrated among tumor cells and in the periphery hepatic lobule (Fig-2 a-c) suggesting that evident serious injury in the paracancerous tissue has taken place, while the TNF-? and IL-10 contents are also elevated significantly.
So, it is presumed that peri-inflammatory factors response may be the principle cause of ischemic hypoxia that occurs in the adjacent tissues after TAE intervention. Therefore, it can be concluded that IR has taken place. Such complicated signaling ensures that, whenever TNF-? is released, various cells with vastly diverse functions and conditions can all respond to inflammation appropriately.Under normal physiological states, NF- ?B is a dimer, mainly distributed in the cytoplasm that is composed of P105, P100, P50, p52, RELA/p65, c-REL and RELB7 proteins, and functions as a transcriptional activator protein factor 35,36. This plays a critical role in the regulation of gene expression of inflammation related to malignant tumors; inhibitors of this pathway are good for cancer treatment. It is activated by several stimuli as inflammatory cytokines, bacteria endotoxins, hypergylcemia and hypoxia activating survival pathway 37.
When the cells are triggered by inflammatory factors such as TNF-? and reactive stimulus such as ischemia, reperfusion injury and, hypoxia etc.- it combines with the inhibitory protein I?B? that normally binds to NF-?B and inhibits its translocation 38. Further, the oxidative phosphorylation of I?B? by I?B kinase (IKK) and is subsequently degraded, NF-?B is unbound from the receptor protein (sequestering), enters the nucleus- negatively regulating the transcritptional activity i.
e inhibiting its translocation 36,38. Angiotensin-II type 1 receptor (AT1) downstream signaling is recognized to lead to the activation of NF-?B 39, thereby regulating the reaction of inflammatory factors and finally producing a protective effect on the body 40. Other studies have found that severe ischemia and anoxia can lead to the excessive activation of NF-?B and enhance the expression of the downstream inflammatory factor TNF-?, which further stimulates the synthesis of IL-10, antagonizing inflammation and anti-inflammatory during IR process. This causes cell necrosis, eventually leading to tissue damage 41.
Similarly, the results of our study show that as compared with the control group and the contrast groups; the positve expression rates of NF-?B was significantly increased in the adjacent tissues of the TAE group. ConclusionWe can conclude that the injury to the paracancerous liver tissue after TAE may be caused by ischemic reperfusion injury i.e hypoxia/anoxia trigerred by intraoperative embolic agents entering the residual liver tissue. This has lead to excessive activation of NF-?B and regulation of the downstream syntheis of inflammatory factors like TNF-? and IL-10 which lead to the inequillibrium of pro-inflammatory and anti-inflammatory factory along the IR process. By inhibiting the over expression of NF-?B, we can reduce the excessive synthesis of TNF-? and IL-10, there by preventing the inequillibrium of pro-inflammatory and anti-inflammatory factors. Our future projects endevour to target NF-?B and research interventional methods with various embolization materials to prevent this damage to residual normal liver tissue and preserve the hepatic function that is degrading which is induced after TAE.
Gaining control over the IR process can improve the survival rate and decrease mortality.DeclarationsEthics approval and consent to participateThis publication and study protocols were approved by the Biomedical Ethics committee of The First Affiliated Hospital of Dali University. Consent to participate is not applicable. Consent to publishNot applicableAvailability of data and materialsThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Competing InterestsThe authors declare that they have no competing interests to disclose.FundingThis work was supported by the National Natural Science Foundation of China (Grant No: 81241131, 81660300); The Department of Medical Imaging and Interventional Surgery, the First Affiliated Hospital of Dali University, key subjects for medical imaging and study. Author ContributionsBV and W-BR contributed to the manuscript by writing the original draft, reviews, editing, conception and design, modelling and sampling, data collection. DW contributed to conceptualization, experimentation, interventional surgical procedures, data curation, reviews. YY-J and WC-H contributed with materials and methods, interventions, post-processing, analysis.
KS contributed to literature reviews, editing, randomization and statistical data compalition. All authors read and approved the final manuscript. AcknowledgementsThe authors would like to thank Dr.
Wang Guang Ming, Department of Pathology and Immune-histochemistry, The First Affiliated Hospital of Dali Uniersity for providing high quality images and expertise in the pathological analysis. The authors would also like to thank Dr. Duan Wen Shuai in conducting and supervising the interventional and DSA procedures. The authors appreciate the support and participation of all the personnel of the Department of Radiology throughout this experiment.
ReferencesSun H, Han B, Zhai H, et al. Significant association between MTHFR C677T polymorphism and hepatocellular carcinoma risk: A meta-analysis. J Tumor Biology. 2014; 35(1): 189-193.
Liu Li, Shao Tian-peng, Yang Tao, Cao Jian-min, Lu Guang—ming, Xu Jian. Intra—arterial infusion of rAd-p53 combined with transcatheter arterial embolization for middle—late stage hepatocellular carcinoma?observation of its therapeutic efficacy. J Intervent Radiol. 2016;(3):210-213. Ma, Ka Wing; Cheung, Tan To. Surgical resection of localized hepatocellular carcinoma: patient selection and special consideration. J Journal of Hepatocellular Carcinoma.
2016;4: 3. Vogl TJ, Straub R, Eichler K, Woitaschek D, Mack MG. Malignant liver tumors treated with MR imaging-guided laser-induced thermotherapy: experience with complications in 899 patients (2,520 lesions). Radiology. 2002; 225:367– 377.Midorikawa T, Kumada K, Kikuchi H et al. Microwave coagulation therapy for hepatocellular carcinoma.
J Hepatobiliary Pancreat Surg. 2000; 7:252–259.Allgaier HP, Deibert P, Zuber I, Olschewski M, Blum HE. Percutaneous radiofrequency interstitial thermal ablation of small hepatocellular carcinoma. Lancet. 1999; 353:1676–1677. Gbolahan, Olumide B.; Schacht, Michael A.
; Beckley, Eric W.; LaRoche, Thomas P.; O’Neil, Bert H.; Pyko, Maximilian. Locoregional and systemic therapy for hepatocellular carcinoma.
J Journal of Gastrointestinal Oncology. 2017;8 (2): 215–228. Liang YJ, Yu H, Feng G, et al.High-Performance Poly(lactic-co-glycolic acid)-Magnetic Microspheres Prepared by Rotating Membrane Emulsification for Transcatheter Arterial Embolization and Magnetic Ablation in VX2 Liver Tumors. J ACS Appl Mater Interfaces. 2017;9(50):43478-43489.
Lu D, Abulimiti A, Wu T, et al. Pulmonary surfactant-associated proteins and inflammatory factors in obstructive sleep apnea. J Sleep and Breathing. 2018; 22(1): 99-107.Epanchintsev A, Shyamsunder P, Verma RS, et al. IL-6, IL-8, MMP-2, MMP-9 are overexpressed in Fanconi anemia cells through an NF-kappaB/TNF-alpha dependent mechanism.
J Mol Carcinog. 2014;54(12):1686–1699.Alda L Tam, Marites P, Joe Ensor, et al.
Rabbit hepatic artery anatomical variations: Implications on experimental design. Acta Radiol. 2014; 55(10):1226-1233. Shope RE, Hurst EW. Infectious papillomatosis of rabbits. J Exp Med. 1933; 58:607–624.Wang D, Bangash AK, Rhee TK, et al.
Monitoring embolization in rabbits with VX2 tumors-transcatheter intraarterial first-pass perfusion MR imaging. Radiology. 2007; 245:130–139.Zhu Xian hai, Wand Wei yu, Shi Chang gao, Qin Han lin, Lu Wei fu.
Temperature-controlled radiofrequency ablation for the treatment of hepatocellular carcinoma VX2-an experimental study. J Intervent Radiol. 2016;(1):56-60. Chen JH, Lin YC, Huang YS, et al. Induction of VX2 carcinoma in rabbit liver: comparison of two inoculation methods. Laboratory Animals.
2004; 38, 79-84. Xu Tao Chen Xiao ping, Li Dan. Expression of EMMPRIN in rabbit VX2 liver tumor tissue and significance. J Chin J Exp Surg.
2007;(1):37-39.Mescher, Anthony L. Junqueira’s Basic Histology text and atlas.
McGraw-Hill Education. 2013, p. 333.
Breedis C, Young G. The blood supply of neoplasms in the liver. Am J Pathol. 1954; 30:969–985. Wang LQ, Persson BG, Bergqvist L, Bengmark S et.al. Influence of dearterialization on the distribution of absolute tumor blood flow between hepatic artery and portal vein.
Cancer. 1994;74:2454–2459.Jaeger HJ, Mehring UM, Castaneda F, Hasse F, Blumhardt G, Loehlein D, Mathias KD.
Sequential transarterial chemoembolization for unresectable advanced hepatocellular carcinoma. Cardiovasc Intervent Radiol. 1996;19:388–396.
Kennedy KA, Rockwell S, Sartorelli AC. Preferential activation of mitomycin C to cytotoxic metabolites by hypoxic tumor cells. Cancer Res.
1980; 40:2356–2360. Pan SS, Andrews PA, Glover CJ, Bachur NR. Reductive activation of mitomycin C and mitomycin C metabolites catalyzed by NADPH-cytochrome P-450 reductase and xanthine oxidase.
J Biol Chem. 1984; 259:959–966. Izumi Miki, Satoru Murata, Fumio Uchiyama, et al. Evaluation of the relationship between hepatocellular carcinoma location and trans arterial chemoembolization efficacy. J World J Gastroenterol. 2017;23(35):6437-6447.Guo Huan qing, Che Chi dan, Yan peng, Li Ren-fei, Li Wei, Yang Po.Transcatheter super-selective hepatic arterial embolization for the treatment of polycystic liver disease: a preliminary study.
J J Intervent Radiol. 2015;24(8):676-679.Sakamoto I, Aso N, Nagaoki K et al. Complications associated with transcatheter arterial embolization for hepatic tumors. Radiographics. 1980; 18:605–619. Linlin Chen, Huidan Deng, Hengmin Cui, et al.
Inflammatory responses and inflammation-associated diseases in organs. J Oncotarget. 2018; 9(6): 7204-7218.Maria C Seleme, Kate Kosmac, Stipan Jonjic, et al. Tumor Necrosis Factor Alpha-Induced Recruitment of Inflammatory Mononuclear Cells Leads to Inflammation and Altered Brain Development in Murine Cytomegalovirus-Infected Newborn Mice. J Journal of Virology. 2017;91(8): e01983-16.Mosser DM, Edwards JP.
Exploring the full spectrum of macrophage activation. Nature Reviews. Immunology. 2008;8 (12): 958–69.Schall TJ, Lewis M, Koller KJ, Lee A, Rice GC, Wong GH, Gatanaga T, Granger GA, Lentz R, Raab H, et al.
Molecular cloning and expression of a receptor for human tumor necrosis factor. Cell. 1990; 61 (2): 361–70.Khoshdel A, Kheiri S, Omidvari P, et al.Association between Interleukin-10-1082 G/A and Tumor Necrosis Factor-? 308 G/A Gene Polymorphisms and Respiratory Distress Syndrome in Iranian Preterm Infants. J Mediators Inflamm. 2017;(1):638-645.Zheng LM, Ojcius DM, Garaud F, Roth C, et al.
Interleukin-10 inhibits tumor metastasis through an NK cell-dependent mechanism. J The Journal of Experimental Medicine. 1996;184 (2): 579–84.
Men T, Yu C, Dan W, et al. The impact of interleukin-10 (IL-10) gene 4 polymorphisms on peripheral blood IL-10 variation and prostate cancer risk based on published studies. J Oncotarget. 2017;8(28):45994-46005.Palacz Wrobel M, Borko wska P, Paul Samojedny M, et al. Effect of apigenin, kaempferol, and resveratrol on the gene expression and protein secretion of tumor necrosis factor alpha (TNF-?) and interleukin-10 (IL-10) in RAW-264.7 macrophages. J Biomedicine ; pharmacotherapy.
2017;93:1205.Zhang Y, Hu F, Wen J, et al. Effects of sevoflurane on NF-?B and TNF-? expression in renal ischemia–reperfusion diabetic rats.
J Inflammation Research. 2017; 66(10): 901-910. Bakhtiari E, Hosseini A, Boroushaki M, et al.
Angiotensin II receptor antagonist olmesartan and NF-kappaB inhibitor as cytotoxic and apoptotic agents in the MCF-7 human cell line. J Journal of Chemotherapy. 2016;28(4), 314-320. Bakhtiari E, Hosseini A, Boroushaki M, et al. Synergistic, cytotoxic and apoptotic activities of olmesartan with NF-?B inhibitor against HeLa human cell line. Toxicol Mech Methods, Early online. 2015;1-8. Kim HJ, Hawke N, Baldwin AS, et al.
NF-kappaB and IKK as therapeutic targets in cancer. J Cell Death Differ. 2006; 13:738–47.Hou J, et al. N-Myc-Interacting Protein Negatively Regulates TNF-alpha-Induced NF-kappaB Transcriptional Activity by Sequestering NF-kappaB/p65 in the Cytoplasm.
J Sci Rep. 2017;7(1): 14579.Nagai N, Izumi-Nagai K, Oike Y, et al. Suppression of diabetes- induced retinal inflammation by blocking the angiotensin II type 1 receptor or its downstream nuclear factor-kB pathway.
J Invest Ophthalmol Vis Sci. 2007;48:4342–50.Li W, Wang X, Niu X, et al.
Protective Effects of Nobiletin Against Endotoxic Shock in Mice Through Inhibiting TNF-?, IL-6, and HMGB1 and Regulating NF-?B Pathway. J Inflammation. 2016;39(2):786-797.
Chandra K, Gogia A, Kakar A, et al. To study co-relation of inflammatory marker TNF-? in diabetic mellitus patients and healthy nondiabetic adults. J Current Medicine Research and Practice. 2016;6(1):8-11.Figure legendsFig. 1 PreparationIsolated VX2 tumor for extraction.Extracted VX2 tumor from the hind leg placed in a sterile tray containing 20000 IU of Gentamycin and normal saline.
Tumor suspension with several pieces (1mm3) tumor material made from thawing extracted hind leg tumor. Fig. 2 Gross pathologyA 1mm3 fragment of VX2 carcinoma was inoculated into the sub-capsule of the left lobe liver of a rabbit.A solitary mass (arrow) about 2 x 2 cm in size developed in the liver 2 weeks after implantation of a fragment of VX2 carcinoma. In addition, multiple small masses were also noted over the liver surface (arrow heads).
Post TAE of the liver with embolized tumor (arrow) and hypoxic ischemic injury with a significant reduction in the residual normal liver tissue after TAE (asterisks). Fig. 3 CT ImagingPlain CT demonstrating a well-defined, homogeneous hypodense mass lesion in the left anterior lobe of the liver comparable to HCC (arrow).Contrast CT (arterial phase) showing peripheral enhancement of the mass lesion (arrow head).
Axial T1 weighted imaging MR sequence demonstrating a mass lesion with behavior comparable to HCC with the hypo-intense signal (arrow). Axial T2-weighted imaging MR sequence showing the mass lesion with the hyper-intense signal (arrow head). Fig. 4 Trans arterial Embolization, left lateral viewUptake of lipiodol after embolizing the principal artery of the tumor (arrow heads) from intervention through the left hepatic artery (arrow).
Embolic material deposits due to residual liver tissue uptake after TAE (arrow heads).Fig. 5 Arteriography Hepatic angiogram, left lateral view confirms dominant hyper vascular mass (arrow heads) arising from the left hepatic artery (arrow) which shows a “Cluster sign”.Enhancement of the complete vascular bundle of the tumor after contrast administration. Fig. 6 Histopathology and Immune-histochemistry (a-c) Coned out sections of liver tissue adjacent to the tumor show a large number of neutrophilic infiltration around the portal area and hepatic lobule; (d-f) the expression of NF-?B in the para-cancerous liver tissue.
TAE group showed significantly higher infiltration and expression than that of other groups. a;d- control group, b;e- contrast group, c;f- TAE group, stained with hematoxylin and eosin (H.E), and Envision double staining method, Magnification x 100.
