Effects Of Silencing The DUSP1 Gene Using Lentiviral Vector-mediated SiRNA On The Release Of Pro-inflammatory Cytokines In Mice With Acute Pancreatitis

BackgroundAcute pancreatitis(AP)is an acute inflammatory disease mainly manifested as acinar cell injury,oxidative stress,and pancreatic inflammation,frequently caused by gallstone disease or excess alcohol ingestion,and is a main cause of morbidity and mortality worldwide.Most patients suffering from AP are in a mild form but about 20%of patients develop a complicated life-threatening disease with a risk of organ failures.The severity of early acute pancreatitis is mainly determined by the presence or absence of organ failure and the duration of organ failure.According to the classification criteria of acute pancreatitis in 2012 Atlanta conference,acute pancreatitis was divided into three types:mild,moderate and severe acute pancreatitis.Patients with mild acute pancreatitis have neither organ dysfunction nor local and systemic complications,and generally have a good prognosis with almost zero case fatality.Patients with moderate severe acute pancreatitis are often accompanied by transient organ failure(which can recover spontaneously within 48 hours),or with local or systemic complications without persistent organ failure.Patients with moderate severe acute pancreatitis have a higher mortality than those with mild acute pancreatitis,but much lower than those with severe acute pancreatitis.Sustained organ dysfunction exists in patients with severe acute pancreatitis(lasting for more than 48 hours),and the mortality rate of severe pancreatitis is 36%to 50%,which is higher if combined with infection.Once appearing sustained organ dysfunction,it is easy to dividing the patients with AP into mild or severe acute pancreatitis,but there is no help on how to improve the prognosis.To improve the prognosis,the progression and results of the disease of acute pancreatitis should be well predicted in the early stages of the disease,so that more effective treatments and intervention measures may be made as early as possible to avoid the occurrence of multiple organ function failure.A technique called RNA interference(RNAi)is a kind of new biological technology in recent years,because it can be simply and effectively implemented.More studies have shown that trace of siRNA application can make the coding content dropped more than 90%of the disease-causing gene product,some even can achieve the result of gene knock out completely.As a powerful tool replacing the gene knockout,RNAi has a broader application prospect in gene function research and gene therapy.Lentivirus vector,a gene therapy vector based on human immunodeficiency virus(HIV),is a commonly used RNA interference technique currently,which can maintain long-term stability in different mammalian cells,and infect both mitotic and non-mitotic cells.Lentiviral vectors have excellent packaging ability,extensive cellular tropism,and have developed a system that can be used to reduce the expression of target genes.Compared with other retroviruses,lentivirus has significant advantages such as being able to infect non-dividing cells,accommodate large fragments of exogenous genes,and express for a long time without pathologic damage.Dual-specificity phosphatase-1(DUSP1),also known as mitogen-activated protein kinase(MAPK)phosphatase 1,is a member of the MAPK phosphatase family and a potent negative regulator of MAPK activity.Its expression is induced by many extracellular stimuli,including growth factors,cytokines and stress.The DUSP1 gene is considered a tumor suppressor and a regulator of cancer-associated inflammation.DUSP1 is also important in anti-inflammation effects.MAPKs are considered evolutionarily well-conserved serine and threonine protein enzymes,which are involved in signal transduction pathways linking cell surface receptors with main regulative nuclear and intracellular targets.MAPKs are considered to be main signal transducers in the early stage of the development of AP.In mammals,there are several MAPK enzymes responsible for cell proliferation,apoptosis,differentiation and survival.MAPK phosphatases,including DUSP1,inhibit signal transduction and cytokine activation via MAPK dephosphorylation or interference with effector molecules binding to MAPKs.The key to the exacerbation or even deterioration of acute pancreatitis is the loss of control over multiple inflammatory mediators during acute pancreatitis,leading to a cascade of reactions that leads to massive necrosis of pancreatic tissue and a complicated condition because of systemic multi-organ failure.In this regard,the role of cytokines has been the focus of attention.This study aims to observe the proinflammatory factor expression and MAPK pathways related gene protein expression in serum and tissues in mice model of acute pancreatitis,and to explore the proinflammatory factor release control mechanism and the effects on multiple organ injury by lentivirus vector silencing DUSP1 to enhance the role of MAPK signal pathway and PD98059(MAPK signal pathway inhibitors)inhibiting the MAPK signaling pathways.ObjectiveThis study aims to observe the proinflammatory factor expression and MAPK pathways related gene protein expression in serum and tissues in mice model of acute pancreatitis,and to explore the proinflammatory factor release control mechanism and the effects on multiple organ injury by lentivirus vector silencing DUSP1 to enhance the role of MAPK signal pathway.Methods1.Construction and detection of the psiRNA-DUSP1 vectorTwo DUSP1-siRNAs and one negative control(NC)sequence were designed according to the mouse DUSP1 gene sequence in the gene bank as follows:siRNA1:5’-TAG CGT CAA GAC ATT TGC TGA-3’;siRNA2:5’-CTG TAC TAT CCT GTA AAT ATA-3’;and negative:5’-AAC TGG ACT TCC AGA AGA ACA-3’.The expression of DU SP1 was detected and siRNA with high silencing efficiency was screened2.Lentivirus packaging and titer determination.The pSIH-DUSP1-siRNA and pSIH-NC with high silencing efficiency were selected to co-transfect 293T cells with a packaging plasmid mixture respectively using the lipofection method,and the virus titer was measured.3.Preparation and grouping of the AP mouse model.A total of 105 male KM mice in healthy condition were used.Prior to the experiment,the mice used for the AP model were fasted for 12 h for gastrointestinal decompression,however,they all had free access to water.The mice were intraperitoneally injected twice with a 1 h interval with 20%L-arginine at 4 g/kg to establish the AP mouse model.Using a Table of random numbers,the mice were divided into seven groups with 15 mice in each group:Control group(mice injected with the same dose of normal saline);siRNA group(mice injected with 0.88 mg/kg DUSP1-siRNA lentivirus);AP group(AP mouse model);AP+PD98059 group[0.5 h pre-induction and 1 h post-induction,the mice were intraperitoneally injected with PD98059 at 10 mg/kg;AP+siRNA group(following successful model establishment,lentiviruses containing 0.88 mg/kg DUSP1-siRNA were injected into mice intraperitoneally);AP+scramble group(following successful model establishment,0.88 mg/kg scramble-siRNA lentiviral vectors were injected intraperitoneally);AP+PD98059+siRNA group(0.5 h pre-induction and 1 h post-induction,mice were intraperitoneally injected with PD98059 at 10 mg/kg,and following successful model establishment,lentiviruses containing 0.88 mg/kg DUSP1-siRNA were injected into mice intraperitoneally).The mice in each group received cardiac blood sampling(as blood specimen)at 12,24 and 48 h post-modeling,respectively.After 48 h,the mice were sacrificed and pancreatic tissue,liver tissue,lung tissue and kidney tissue were immediately obtained.4.Elisa was used to measure the proinflammatory factors,HMGB1 and S100A12 in serum.At 12,24 and48 h post-model establishment,five mice in each group were selected for cardiac blood sampling.Elisa was used to measure the proinflammatory factors,HMGB1 and S100A12 in serum,Strictly following the kit requirements.5.Determination of serum amylase and lipase levels.The levels of amylase and lipase in serum were determined at 12,24 and 48 h post-model establishment in each group.The test of each index was performed strictly in accordance with the requirements of the reagent package6.Hematoxylin and eosin(HE)staining.The pancreatic,liver,kidney and lung tissues of mice in each group were removed and fixed with 4%formaldehyde,and immersed and embedded in paraffin.The paraffin-embedded pancreatic tissues were cut into sections,Using the morphological image analysis system,images of the HE-stained sections of pancreatic,liver,kidney and lung tissues in each group were captured.7.Reverse transcription-quantitative polymerase chain reaction(RT-qPCR)analysis.Based on the manufacturer’s protocol of the TRIzol kit,total RNA from the tissue specimens was extracted via the TRIzol one-step method.The quality and concentration of total RNA were identified and determined.The reverse transcription of the extracted RNA was performed in two steps according to the protocol of the kits.The RT-qPCR was performed using the TaqMan probe method according to the kit protocol to measure the mRNA expressions of pro-inflammatory factors(TNF-a,IL-1β and IL-6),DUSP1,HMGB1,and S100A12 in various organs.8.Western blot analysis.Pancreatic,liver,kidney and lung tissue samples were extracted and shattered on ice using ultrasound,following removal of blood and other tissues.Proteins related to the DUSP1 and MAPK pathways were detected by western blotting.Results1.The siRNA2 sequence was designed with high silencing efficiency,and the infection rate of lentivirus packaged by pSIH-DUSP1-siRNA2 to 293T cells was over 90%.2.Compared with the control group,the serum levels of amylase and lipase of mice in the siRNA group were increased as well as pro-inflammatory factors,HMGB1 and S100A12 in serum and tissues,but there was no significant difference.In the other 5 groups,the expression of each index increased significantly.3.Compared with the AP group,the expression of DUSP1 in the AP + siRNA group was decreased,while the expression of various indicators in the serum was significantly increased,and the expression of TNF-α,IL-1β,IL-6,HMGB1 and S100A12 mRNA in the tissue,as well as the related gene proteins p-ERK、p-JNK and p-p38 in the MAPK signaling pathway was also significantly increased,and the pathological changes in the pancreatic tissue were aggravated.In the AP + PD98059 group,the expression of various indexes in serum decreased,and the expression of TNF-a,IL-1β IL-6,HMGB1 and S100A12 mRNA in tissues as well as the related gene proteins p-ERK、p-JNK and p-p38 in MAPK signaling pathway also decreased,while the degree of edema in pancreatic tissues decreased.There was no significant difference between AP+scramble group and AP+PD98059+siRNA group.4.In the mice with acute pancreatitis in each group,the expression levels of HMGB1 and S100A12 in serum were positively correlated with the expression levels of TNF-α,IL-1βand IL-6.5.Compared with the AP group,the pathological changes of pancreas,liver,kidney and lung tissues in the AP + siRNA group were significantly aggravated,while the pathological changes in the AP + PD98059 group were reduced.Conclusion1.The expression of DUSP1 in the cells was effectively inhibited by lentivirus packed with pSIH-DUSP1-siRNA2.2.In the mice model with acute pancreatitis,silencing DUSP1 gene can promote the release of pro-inflammatory cytokines.Its mechanism may be that it plays its active role through phosphoiylation of ERK,JNK and p38 in MAPK signaling pathway,enhancing the effect of MAPK signaling pathway,thus promoting the release of pro-inflammatory cytokines.3.The expression levels of HMGB1 and S100A12 in serum are positively correlated with the expression levels of TNF-α,IL-1β and IL-6,suggesting that there is a positive feedback relationship between the pro-inflammatory cytokines(TNF-α,IL-1β and IL-6)and HMGBI and S100A12,the upstream factors of MAPK signaling pathway,jointly promoting the development of inflammatory processes.4.Overexpression of TNF-α,IL-1β,IL-6,HMGB1 and S100A12 can aggravate the pathological changes of pancreas,liver,kidney and lung tissues in the mice model with acute pancreatitis,making multiple organ failure easier and aggravating the condition of acute pancreatitis.5.Early detection of DUSP1 expression can predict the probability of multiple organ failure in advance.By regulating the DUSP1 and MAPK signaling pathways,the degree of organ damage in acute pancreatitis mice can be changed,providing a new target for treatment.