TAB3 overexpression promotes NF-κB activation and inflammation in acute pancreatitis.

Am J Blood Res 2020;10(4):118-123 www.AJBlood.us /ISSN:2160-1992/AJBR0115283 Original Article TAB3 overexpression promotes NF-κB activation and inflammation in acute pancreatitis Lili Gao1, Hongmei Zhao1, Hong Sun1, Ying Huang2 Department of Emergency, The Affiliated Huai’an No. 1 People’s Hospital of Nanjing Medical University, Huai’an, Jiangsu Province, China; 2Department of Intensive Care Unit, The Affiliated Huai’an No. 1 People’s Hospital of Nanjing Medical University, Huai’an, Jiangsu Province, China 1 Received May 29, 2020; Accepted June 24, 2020; Epub August 25, 2020; Published August 30, 2020 Abstract: Acute pancreatitis (AP) is a clinically common inflammatory disease, NF-κB activation and the secretion of pro-inflammatory mediators have been considered as the main events of AP. According to reports, TAB3 is essential for NF-κB activation and participates in inflammatory responses. In this study, we used caerulein to establish an AP rat model and cell model. The expression of TAB3 was measured both in control group and AP group by Western blot and Immunohistochemistry. We firstly found the expression of TAB3 was significantly increased in caerulein-induced AP rat and cell model compared with control group, especially at 8 h. Furthermore, the increasing expression of TAB3 in AP group was also accompanied by increased levels of pro-inflammatory mediators (TNF-α, IL-6 and LDH). In addition, the decreasing expression of TAB3 in TAB3-siRNA transfected AP AR42J cells was accompanied by reduced levels of pro-inflammatory cytokines and inhibited the production of p-P65. These findings suggested that TAB3 may accelerate the inflammatory responses of AP through NF-κB activation. Keywords: TAB3, NF-κB, AR42J cells, acute pancreatitis, inflammation, caerulein Introduction Acute pancreatitis is a common clinical condition with higher incidence and mortality [1-3]. The generally accepted pathogenesis is the activation of trypsinogen, which could provoke pancreatic auto-digestion and subsequently systemic inflammatory response [4]. Nevertheless, its precise pathogenesis has not yet been fully elucidated. Several animal models have been used to study the pathogenesis of acute pancreatitis, one of the most common models is caerulein-induced AP model [5-8]. Caerulein is a gastric regulatory molecule similar to cholecystokinin, which could stimulate the secretion of pancreas. The mechanisms involved in are NF-κB activation and pro-inflammatory response. According to many related literatures, TAB3 (transforming growth factor β-activated kinase binding protein 3) is a binding protein of TAK1 and can link TAK1 to TRAFs, subsequently activating NF-κB pathway. TAK1 first activates IKKs, subsequently IKKs phosphorylate IκBs and lead to the activation of NF-κB [9-14]. In this study, we found that TAB3 was overexpressed in AP. However, its molecular mechanism is not clear. In this study, we established a caerulein-induced AP cell and animal model to analyze the expression level of TAB3 and to evaluate its potential effect on pro-inflammatory cytokines. Furthermore, we explored the role of TAB3 in NF-κB activation. Since NF-κB activation has a positive correlation with the inflammatory responses of AP, so we speculated that TAB3 may participate in AP through NF-κB pathway. To test our hypothesis, we transfected TAB3-siRNA into AP AR42J cells and found TAB3 siRNA could inhibit NF-κB activation in caerulein-induced AP, as well as reduce the levels of inflammatory mediators. Our study firstly demonstrated that the upregulation of TAB3 accelerated the AP progression via facilitating NF-κB activation. This study may provide a new clinical treatment direction for AP. TAB3 overexpression in acute pancreatitis Materials and methods Cell culture Rat pancreatic AR42J acinar cells were purchased from ATCC and maintained at 37°C in humidified air with 5% CO2 and 95% oxygen. The 100 nM caerulein (Sigma, #C9026) and PBS (phosphate buffered saline) was respectively added to the caerulein group and the control group at different time (0-24 h). ly dehydrated, cleared, covered and examined under a Leicalight microscope. Animal model SD male rats (weighing from 180 g to 200 g) were purchased. 50 μg/kg caerulein was injected into the abdominal cavity of SD rats every four hours to induce AP model. Meanwhile the control groups were treated with PBS. Finally, all treated rats were killed from 4 h to 24 h. Western blot analysis Enzyme linked immunosorbent assay The protein samples were analyzed by PAGE and transferred to a PVDF membrane. The PVDF membrane was firstly blocked with 5% skim milk and then incubated with antibody against TAB3 (1:500, Santa Cruz Biotechnology, USA), IKKβ (1:500, Cell Signaling), P65 (1:500, Santa Cruz), p-P65 (1:500, Santa Cruz), β-actin (1:1000, Santa Cruz). After washing with TBST, the transfer membrane was placed into the second antibody and incubated for 2 h in the dark. Subsequently, ECL system (enhanced chemiluminescence system, Thermo Scientific Pierce, Rockford, USA) was used to detect the immunocomplexes, and the gray value of bands was measured by Bio-Rad image processing system. Finally, the density of bands was compared by ImageJ (NIH). After treatment with caerulein, the supernatant fluid was collected. We use ELISA kits to measure the level of pro-inflammatory mediators in the supernatant fluid. siRNA and transfection To study the level of TAB3, an AP rat model was established [15]. We performed immunohistochemistry analysis to study the expression of TAB3 in normal tissues and caerulein-induced pancreatitis tissues. As Figure 1A demonstrated, the TAB3 expression level was obviously increased in AP group compared with the control. Next, Western blot analyses was carried out to explore the expression of TAB3 in control and caerulein-induced pancreatitis tissues. As expected, the expression of TAB3 was significantly upregulated in AP group compared with the control, especially at 8 h (Figure 1B, 1C). Thus, further experiment was required to clarify the possible function of TAB3 in the occurrence and development of AP. The TAB3 siRNA target sequence was as follows: 5’-CTGAGGAAATGACAAGATT-3’ (siRNA#1), 5’-GGTTGAAGTCTGAAGTTAA-3’ (siRNA#2), 5’TCCTTCATACATGCACATA-3’ (siRNA#3). AR42J cells were planted in dishes and replaced with fresh culture for transfection siRNA. The 100 nM caerulein was added to AR42J cells after transfection for 40 h and were collected to carry on Western blot after transfection for 48 h. Immunohistochemistry Firstly, the rat pancreas specimens were respectively fixed and embedded by 10% formalin and paraffin wax, then all sections were successively dewaxed, rehydrated, and processed in citrate buffer for antigen retrieval. Subsequently, the slices were successively treated with 3% H2O2 and antibody against TAB3. After cleaning with PBS, the slices were incubated with DAB. Finally, the sections were respective119 Statistical analysis The (...truncated)