The Mechanism Of Daphnetin To Improve Lung Injury Associated With Severe Acute Pancreatitis In Mice By Inhibiting JAK2-STAT3 Pathway

Background and PurposeAcute pancreatitis can be regarded as an inflammatory response of the pancreas,often with suddenly onset and highly mortality.It can often cause severe acute pancreatitis(SAP),which can cause damage to many related organs in the body,among which the lungs are the most vulnerable organ to be affected.Despite considerable time and effort devoted to research into severe acute pancreatitis associated lung injury(PALI),there is still a lack of effective treatment.Therefore,it is necessary to explore the molecular mechanisms of PALI in order to develop new potential therapies.The JAK-STAT signaling pathway plays an indispensable role in regulating many cellular processes such as apoptosis,inflammatory response and oxidative stress response.This pathway can be activated by numerous mediators,such as cytokines,growth factors,and proinflammatory mediators.When these mediators bind to the corresponding receptors,they can promote the phosphorylation of JAK,and the phosphorylated JAK protein can further activate the STAT protein.The activated protein is transformed into p-STAT form,which can be translocated to the nucleus and regulate the transcription of receptor genes.Evidence from many clinical and preclinical studies suggests that the JAK2-STAT3 pathway plays a key role in pancreatitis and lung injury.However,little is known about the role of this pathway in PALI progression.Daphnetin(7,8-dihydroxycoumarin)is a natural coumarin derivative that has been reported to have a variety of pharmacological effects,such as pain relief,inhibition of inflammatory response,and antioxidant.At present,the drug has been applied in many diseases in clinic.However,no studies have been conducted to elucidate the effect of daphnetin on PALI and its possible mechanism.The purpose of this study was to investigate whether daphnetin can exert its protective effect in L-arginine-induced PALI mouse model,and whether the mechanism can exert its protective effect through the JAK2/STAT3 signaling pathway.MethodsTwenty-four C57BL/6 male mice were randomly divided into 4 groups:CON group,SAP group,DAP group and SAP+DAP group.The model of severe pancreatitis associated lung injury was established by intraperitoneal injection of L-arginine twice(1 h interval),and mice in the DAP treatment group were pretreated by intraperitoneal injection of DAP 30min before modeling.24 h after induction,blood and tissue samples were collected.H&E staining was performed on the pancreatic tissue of mice to compare the damage of the pancreatic tissue of mice in each group,and pathological score was made with reference to the literature.The serum expression levels of TNF-α,amylase,IL-6 and lipase in each group were detected by ELISA assay,and then the results were analyzed.H&E staining was performed on the lung tissue of mice to compare the pathological damage of the lung tissue of mice in each group.The lung tissues of each group of mice were homogenized by ELISA,and the concentrations of TNF-α,IL-6 and MPO were detected,and the results were statistically analyzed.The expression of macrophages(CD11b)and neutrophils(LY6G)in lung tissues of mice in each group was detected by immunofluorescence.The protective effect of daphnetin on apoptosis of lung cells was studied by TUNEL staining.Western Blot was used to detect the expression levels of JAK2,phosphorylated JAK2(p-JAK2),STAT3 and phosphorylated STAT3(p-STAT3)in lung tissues of mice in each group,and semi-quantitative analysis was performed.The expressions of p-JAK2 and p-STAT3 in lung tissues of mice in each group were analyzed by immunohistochemistry,and semi-quantitative comparative analysis was conducted.Results1.Compared with CON group and DAP group,SAP group showed pancreatic tissue injury,including interstitial edema,massive neutrophils infiltration and necrosis,while daphnein preconditioning group significantly reduced pancreatic tissue injury.2.Compared with CON group and DAP group,serum TNF-α,amylase,IL-6 and lipase levels in SAP group were significantly increased,and serum TNF-α,amylase,IL-6 and lipase levels were decreased after daphnein pretreatment.3.Compared with CON group and DAP group,H&E staining of lung tissue of mice in SAP group showed that alveolar structure was destroyed and surrounded by numerous inflammatory cells,while the change of alveolar injury in SAP+DAP group was significantly less.4.Compared with CON group and DAP group,the levels of TNF-α,IL-6 and MPO in lung of mice in SAP group were significantly increased.The levels of inflammatory cytokines in lung tissues of mice pretreated with daphnetin before SAP induction were significantly reduced.5.Compared with the CON group and the DAP group,immunofluorescence showed that the number of macrophages and neutrophils in the lung tissue sections of the SAP group increased,but daphnetin pretreatment could significantly reduce the number of macrophages and neutrophils.6.Compared with CON group and DAP group,the apoptotic cells were more abundant in the lung tissue of mice in SAP group,but the degree of apoptosis was significantly decreased after daphnein pretreatment.7.Compared with CON group and DAP group,the expression of p-JAK2 and p-STAT3 in SAP group increased,while the contents of p-JAK2 and p-STAT3 in daphnetin pretreated tissue decreased.8.The results of immunohistochemical staining and Western Blot analysis of p-JAK2 and p-STAT3 were the same.Compared with the CON group and DAP group,the expression of p-JAK2 and p-STAT3 in lung tissue of SAP+DAP group was significantly lower than that of SAP group.Conclusions1.Daphnetin preconditioning alleviated pancreatic tissue damage in the mouse model of severe acute pancreatitis.2.Daphnetin preconditioning alleviated lung tissue injury in the mouse model of severe acute pancreatitis.3.Daphnetin preconditioning reduced lung tissue apoptosis in mice with severe acute pancreatitis.4.Daphnetin pretreatment can inhibit the activation of JAK2-STAT3 signaling pathway in lung tissue,and it is speculated that the protective effect of daphnein on lung tissue injury may be through the inhibition of JAK2-STAT3 signaling pathway.