CHEN Jin-feng, MENG Nuo, LEI Yu, FENG Yong, LI Wang-jian, TANG Xi-ping

1.Guangxi Medical University, Nanning 530200, China

2.Guangxi Medical University Cancer Hospital, Nanning 530021, China

Keywords:

ABSTRACT

1.Introduction

Acute pancreatitis (AP) is a common digestive tract disease in the world.When developed into severe acute pancreatitis, patients with systemic multiple organ dysfunction, clinical treatment is difficult,and the mortality rate can be as high as 30% [1,2].Inflammatory response and oxidative stress play an important role in regulating the pathogenesis of AP, and jointly participate in different stages of AP through different pathophysiological mechanisms, thus resulting in damage to the pancreas and other organs of the body[3].Oxidative stress is closely related to the severity of AP disease progression.The progression of AP can increase the intracellular reactive oxygen species (ROS) production and reduce the expression of the antioxidant factor superoxide dismutase (SOD), abnormal SOD levels in turn lead to elevated oxygen free radical levels and induce an excessive oxidative stress response, and inhibition of this response effectively reduces the inflammatory response and alleviates the progression of AP[4-6].At the same time, ROS can be used as a molecular trigger of the pancreatic inflammatory process,and one of the key links of the inflammatory response is the NODlike receptor protein 3 (NLRP3) inflammasome.Studies have shown that the activation of the NLRP3 inflammasome is closely related to the production of ROS [7-10].Therefore, the inhibition of the ROSNLRP3 signaling pathway may be important for the attenuated oxidative stress and inflammatory response damage in acute pancreatitis.In this study, we constructed the rat model of mild and severe acute pancreatitis to explore the protective effect of inhibiting ROS-NLRP3 signaling pathway and the relevant molecular mechanism of the injury of acute pancreatitis, in order to provide some reference for the treatment of acute pancreatitis.

2.Materials and methods

2.1 Materials

2.1.1 Experimental animal

Thirty-six 8-week-old SPF male SD rats weighing 250～280 g were purchased from the Animal Laboratory Center of Guangxi Medical University.Use License number: SYXK (Guangxi) 2020-0004, and Production License number for experimental animals:SCXK (Guangxi) 2020-003.Rats were kept under clean and quiet conditions, moved freely, cleaned their cages regularly, and added feed and drinking water on time.

2.1.2 Drugs and Reagents

Caerulin (Sigma Company, USA), sodium taurocholate (TCI Company, Japan), NAC (Shanghai Biyun Yuntian Biotechnology Company), Adventitia collagenase (Beijing Solaibao Technology Co., Ltd.), DHE fluorescent probe (Beijing Plilai Company),superoxide dismutase (SOD) kit (Nanjing Jiancheng Institute of Bioengineering), HE staining kit, NLRP3 antibody, fluorescence quantitative PCR kit (Wuhan Sevier Biotechnology Co., Ltd.), Rat IL-1β and TNF- α ELISA kit (Hangzhou Lianke Biotechnology Co.,LTD.).

2.1.3 Primary instrument

Microinjection pump (Shanghai Lane Medical Device Co.,Ltd.), Optical microscope (Olympus, Japan), IX51 fluorescence microscope (Olympus, Japan), High-speed refrigerated centrifuge(Eppendorf, Germany), fluorescence quantitative PCR instrument(Bio-rad, USA), ultra spectrophotometer (Thermo Fisher, USA),Multi-function microplate reader (Thermo Fisher Corporation,USA).

2.2 Methods

2.2.1 Animal model establishment

Thirty-six SD rats were randomly divided into 6 groups with 6 rats in each group.The rats were fasted and watered 12 h before modeling.The rats in NC group were not treated.Preparation of AP model: rats were injected intraperitoneally with 50 μg/kg caerulein at an interval of 1 h for 7 times.The rats in the AP+NAC intervention group were injected with 0.2 mL/100 g NAC 30 min before the establishment of the model, and the follow-up operation was the same as that in the AP group.Preparation of SAP model: after 10%chloral hydrate anesthesia, the rats were fixed and disinfected, and the midline incision of the upper abdomen was taken to expose the pancreas, pancreatic duct and duodenum, and then the model was established by retrograde injection of 0.1 mL/min sodium taurocholate (0.1 mL/100 g) into the cholangiopancreatic duct.After the injection, the infusion tube was clamped for about 2 min, and the pancreatic edema and congestion could be seen, which proved the success of the model.The duodenum and pancreas were returned in situ and closed layer by layer.The rats in the SAP+NAC intervention group were injected intraperitoneally with 0.2 mL/100 g NAC, 30 min before the establishment of the model, and the other operations were the same as those in the SAP group.In the SO group, rats gently turned the duodenum and pancreas only after open abdomen and remained in situ, closed the abdomen layer by layer.After surgery, normal saline (20 mL/kg) was injected subcutaneously for fluid replacement, and the rats still fasted and watered after operation.

2.2.2 Collection of specimens

The rats in each groups were anesthetized with 10% chloral hydrate 24 h after the mold creation, and the gross pathology of the abdominal cavity and pancreas was observed by laparotomy, blood was collected from the abdominal aorta, centrifuged at 3000 r/min for 10 min, collected serum and stored in -80 ℃ refrigerator.Part of pancreas and kidney tissue was fixed with 4% paraformaldehyde for paraffin sections for HE staining and immunohistochemical detection; and the rest was kept in -80 ℃ refrigerator.

2.2.3 Pancreatic histopathological score

Pancreatic tissue fixed with 4% paraformaldehyde was routinely embedded in paraffin, sectioned, and stained with hematoxylineosin (hematoxylin-eosin, HE).Pancreatic tissue morphology was visualized under a light microscope and scored with pathology.Pancreatic histopathological scoring criteria are shown in Table 1[11].

Tab1 Histopathological scoring criteria for acute pancreatitis

2.2.4 Single-cell suspension preparation and intracellular ROS concentration detection

Fresh pancreatic tissue was harvested, washed with saline, cut,centrifuged, removed from supernatant, digested with type 1 mg/mL collagenase, 70 μm cell filter by filtration, centrifuged, and red blood cell lysate was added to split on ice for 5 min, PBS buffer was washed and centrifuged and resuspended.The DHE fluorescent probes were added according to the instructions.Intracellular ROS concentrations were visualized under an inverted fluorescence microscope and photographed.

2.2.5 Serum SOD viability determination

Rats were frozen serum, thawed on ice and tested according to the instructions of SOD kit.

2.2.6 Immunohistochemistry staining

Pancreatic tissue sections were routinely dewaxed, blocked,and incubated overnight with NLRP3 primary antibody at 4℃, secondary antibody at room temperature, colored by DAB,counterstained with hematoxylin, and sealed.Pathology was assessed using double-blind film reading and using semi-quantitative criteria[12].

2.2.7 Quantitative polumerase chain reaction(qPCR)

NLRP3 mRNA expression levels in the kidneys of SO, SAP and SAP + NAC groups, with GAPDH as the reference gene, and qPCR results were analyzed by relative quantification of 2-ΔΔCT.Thesequences of the primers used are shown in Table 2.

Tab2 Primer sequences for q PCR

2.2.8 Enzyme linked immunosorbent assay (ELISA)Serum samples of -80 ℃ frozen rats were taken, thawed on ice, and tested on the levels of IL-1 β and TNF- α in rat serum according to the ELISA kit instructions.

2.3 Statistical processing

Data analysis was performed using the SPSS 26.0 and GraphPad Prism 8.0 statistical software.Measurement data are expressed as mean ± standard deviation, and multiple group comparisons were analyzed by one-way ANOVA.P＜0.05 was considered as a statistically significant difference.

3.Results

3.1 Histopathological changes in the pancreas

There were no significant changes in the pancreatic tissue of rats in the NC and SO groups.Compared with the NC group, the AP rats showed significant edema, but no obvious necrosis, and the AP + NAC group compared with the AP group.Compared with the SO group, the pancreatic tissue of the SAP model group showed obvious congestion and edema, showing extensive inflammatory cell infiltration and cell necrosis; compared with the SAP group, the SAP+ NAC intervention group was significantly improved.See Figure 1.

Fig1 Histopathology of pancreas in each group (HE, ×200)

3.2 Comparison of the ROS levels in the pancreas

Compared with the NC group, the ROS level in the AP group was higher (P＜0.05), and the AP + NAC intervention group was lower than that in the AP group (P＜0.05).The ROS level was significantly higher compared with the SO group in the SAP group (P＜0.05),and the ROS level in the SAP + NAC intervention group decreased compared with the SAP group (P＜0.05).See Figure 2 and Table 3.

3.3 Changes of serum SOD level in each group

SOD activity was decreased in AP group compared with NC group,and SOD activity was increased in AP + NAC intervention group and AP group compared with AP group (all P＜0.05).SOD activity decreased significantly in SAP group and SO group compared with SAP group.Compared with SAP + NAC intervention group, it increased SOD vitality, and decreased oxidative stress (all P＜0.05).See Table 3.

Fig2 ROS fluorescence intensity changes in pancreatic tissue of each group(×100)

Tab3 Comparison of SOD and ROS levels in rats of each group(n =6,)

Tab3 Comparison of SOD and ROS levels in rats of each group(n =6,)

Note: Compared with the NC group, aP＜0.05, compared with the AP group,bP＜0.05; compared with the SO group, cP＜0.05, compared with the SAP group, dP ＜0.05.

Group ROS SOD(U/mL)NC 0.045±0.037 242.950±13.713 AP 0.500±0.067a160.187±7.733aAP+NAC 0.120±0.017b229.870±32.412bF 86.687 13.721 P 0.000 0.006 SO 0.097±0.135 255.080±38.703 SAP 2.354±0.423c107.340±15.646cSAP+NAC 0.303±0.061d201.843±22.523dF 69.755 22.394 P 0.000 0.002

3.4 NLRP3 protein expression in pancreatic tissues of rats was determined by immunohistochemistry

Compared with the NC group, NLRP3 protein expression increased in the AP group, and NLRP3 protein expression decreased in the AP+ NAC intervention group compared with the AP group, which was statistically significant (all P＜0.05); compared with the SO group,NLRP3 protein expression was significantly increased in the SAP group, and NLRP3 protein expression was decreased in the NLRP3 intervention group compared with the SAP group, which was statistically significant (all P＜0.05).See Table 4 and Figure 3.

Tab4 Comparison of NLRP3 IHC staining scores of rat pancreatic tissues in each group(n =6,)

Tab4 Comparison of NLRP3 IHC staining scores of rat pancreatic tissues in each group(n =6,)

Note: Compared with the NC group, aP＜0.05, compared with the AP group,bP＜0.05; compared with the SO group, cP＜0.05, compared with the SAP group, dP ＜0.05.

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3.5 NLRP3 mRNA level determination in kidney tissues

The qPCR results showed that the renal NLRP3 mRNA levels were significantly higher in the SAP model group when compared with the SO group (P＜0.05), and were decreased in the NAC pretreatment group compared to the SAP model group (P＜0.05).See Table 5.

Tab5Relative NLRP3 mRNA expression levels in the rat kidney(n =6, )

Tab5Relative NLRP3 mRNA expression levels in the rat kidney(n =6, )

Note: Compared with the SO group, cP＜0.05, compared with the SAP group,dP ＜0.05.

Group NLRP3 F P SO 1.000±0.000 37.834 0.000 SAP 5.034±0.414cSAP+NAC 1.754±0.961d

3.6 The IL-1β and TNF-α levels in the serum were determined by ELISA

The ELISA results showed that there were higher serum levels of IL-1β and TNF-α in rats in the AP model and SAP model groups than in the corresponding control group.The levels of IL-1β and TNF- were reduced in the NAC pretreatment group as compared with the model group (all P＜0.05).See Table 6.

Tab6Comparison of IL-1β and TNF- α levels in the serum of rats in each group(n=6, )

Tab6Comparison of IL-1β and TNF- α levels in the serum of rats in each group(n=6, )

Note: Compared with the NC group, aP＜0.05, compared with the AP group,bP＜0.05; compared with the SO group, cP＜0.05, compared with the SAP group, dP ＜0.05.

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Fig3 Expression of NLRP3 in pancreatic tissue of each group (×200)

4.Discussion

The pathogenesis of acute pancreatitis is complex.When progressing into severe acute pancreatitis(SAP), it can trigger an inflammatory cascade, which can lead to extrapancreatic organ failure and even death[13].Oxidative stress and inflammatory response play an important role in the occurrence and development of acute pancreatitis.Although the study on the pathogenesis of acute pancreatitis has made great progress in recent years, the association of oxidative stress and inflammatory response in acute pancreatitis still needs to be further explored[11].Therefore, in this study, the retrograde injection of R-F peptide and the rat S AP model to construct the rat SAP model investigated the role of ROS-NLRP3 signaling in the animal model of acute pancreatitis with different severity.

As a core protein of the NLRP3 inflammasome pathway, NLRP3 participates in the immune and inflammatory responses of the body.Studies have shown that increased ROS generation can activate the NLRP3 inflammasome, and subsequently increase the secretion of IL-1β[14,15].However, the proinflammatory cytokines IL-1β and TNF-α are important cytokines in the early inflammatory response in acute pancreatitis, and can activate downstream signal transduction pathways to produce a large number of inflammatory mediators and lead to a sustained inflammatory response[16].The results of this study showed that ROS levels, NLRP3 protein expression, and serum IL-1β and TNF-α levels in rat AP and SAP models compared with the control group (P＜0.05), suggesting that NLRP3 bodies may be activated by ROS generation, which could lead to inflammation and oxidative stress response and aggravate pancreatic injury.

N-acetylcysteine (NAC) is an antioxidant and reactive oxygen free radical scavengers in vivo, with a strong ROS clearance capacity[17].In this study, NAC intervention in rats before acute pancreatitis showed that NAC pretreated rats had decreased inflammatory cell infiltration, reduced cell necrosis changes, and significantly decreased pancreatic ROS levels, NLRP3 protein expression, serum IL-1 β and TNF- α levels (P＜0.05), The antioxidant enzyme SOD activity was significantly increased (P＜0.05), suggesting that ROS expression can be regulated by inhibiting NLRP3 expression in acute pancreatitis, furthermore, it inhibited the secretion of inflammatory factors IL-1β and TNF- α, and alleviated pancreatic tissue oxidative stress and inflammatory injury in rats with acute pancreatitis.Since SAP is often complicated with multiple organ injuries, the kidney is one of the main damaged organs of SAP outside the pancreas, and it is often used as an indicator to evaluate the prognosis[18].Therefore,this study also detected the NLRP3 mRNA expression level of kidney tissue in SO group, SAP model group and SAP + NAC pretreatment group, and found that the NLRP3 mRNA expression level of SAP model group was increased compared with the control group (P＜0.05), NLRP3 mRNA expression of NAC pretreated rats decreased compared with the model group (P＜0.05).It is speculated that the inflammatory level expression of kidney tissue in the disease of rats with severe pancreatitis may be downregulated by inhibiting ROS-NLRP3 signaling, and then the kidney injury secondary to rats with severe pancreatitis may be improved.

In conclusion, the inhibition of ROS-NLRP3 signaling can reduce the oxidative stress and inflammatory injury in pancreatic tissue in different severity of rat models of acute pancreatitis, and may alleviate the secondary kidney injury in rats with severe acute pancreatitis, providing new thinking and experimental basis for the treatment of acute pancreatitis, but there are some experimental evidence of the protection.

Author’s contribution:

Chen Jin-feng: experiment design and operation, data collation and statistical analysis, paper writing; Meng Nuo, Lei Yu, Feng Yong, Li Wang-jian: experimental operation assistant; Tang Xi-ping: research guidance, paper revision, financial support.All authors declare no conflict of interest.