Therapeutic Effect Of Artesunate On Acute Animal Pancreatitis Models And Its Molecular Mechanisms

Background：Acute pancreatitis is a common disease which is initiated from local lesions ofpancreatic tissue to systematic inflammatory response syndrome(SIRS). In severe acutepancreatitis, morbidity and mortality remains high and is ultimately leads to sepsis, septicshock, multi-organ failure and even death. Moreover, the etiology, pathogenesis andpathophysiology of acute pancreatitis remain unclear. The mortality reached5％～13.6％even the clinical use of combination therapy for treatment. Therefore, it is important todevelop useful drug or improved treatment strategies.Object：SIRS is short for systemic inflammatory response syndrome which is caused by avariety of reasons, while sepsis is one of SIRS caused by infection, also known as SIRSsubtype. Given SIRS is throughout the pathophysiology of acute pancreatitis, SIRS shouldbe effectively controlled at the early stage, otherwise, the risk of sepsis, septic shock andmultiple-organ failure will be greatly increased at the late stage. And the mortality will beincreased by4to5times. Therefore, the acute pancreatitis always considered as SIRS andsepsis.Artesunate (AS) is a well-known anti-malarial drug. Our previous studies havereported that AS plays an important role in protecting sepsis mice by decreasing the level ofserum LPS (lipopolysaccharide), TNF-α and IL-6. Given the acute pancreatitis alwaysconsidered as SIRS and sepsis while AS has anti-inflammatory effect. Therefore, using themouse and rat models of acute pancreatitis induced by combination of caerulein and LPS,we explored the therapeutic effects and its molecular mechanisms of artesunate on acutepancreatitis model.Methods：1. The therapeutic effect of AS on acute pancreatitis mouse model⑴The establishment of acute pancreatitis mouse model The caerulein (100μg/kg) was intraperitoneal injected into animals every one hour forsix times, then intraperitoneal injections of LPS (10mg/kg).⑵The therapeutic effect ofAS on acute pancreatitis mice modelSixty Kunming mice were randomly divided into five groups (12per group): normalsaline, AP induction group, AS treatment group which divided into three sub-groupsaccording to the concentration of AS (AS5.0mg/kg, AS2.5mg/kg, AS1.5mg/kg).The normal group was given saline, different concentration of AS was administeredintramuscularly at0and4hours after administration of caerulein in mice as AS treatmentgroup. Mice of AP induction group were received the same volume of saline after inductionof pancreatitis while mice of control group were only received equal saline withoutcaerulein. The animals were sacrificed at18h after the first intraperitoneal injection.⑴General observation of morphology of the pancreas and surrounding tissues;⑵Calculationof the pancreatic histopathology score: HE staining was performed to evaluate themorphologic changes in animal pancreats, and the pancreatic histopathology score wascalculated by double blind method and Rongione method;⑶Pancreatic coefficient wascalculated by the equation: pancreatic coefficient=wet weight of pancreas (mg)/wholemouse weight (g);⑷The levels of malondialdehyde and amylase and the activity of lipase,trypsin and superoxide dismutase (SOD) were determined by commercial kits. The datawere analyzed by SPSS16.0.2. The therapeutic effect of AS on severe acute pancreatitis rat model⑴The establishment of severe acute pancreatitis rat model (SAP)The3.5%sodium taurocholate was retrograde injected through pancreaticobiliary ductinto rats to establish severe acute pancreatitis rat model.⑵The therapeutic effects ofAS on SAP rat modelForty-eight of Kunming mice were randomly divided into five groups (6per group):normal group,, sham operation group, positive group (treated with ulinastatin), SAP group(sodium taurocholate was retrograde injected into biliopancreatic duct) and SAP+AS group(AS was administered in different dosage after the SAP induction at every24-hour for3days). The animals were sacrificed after3days of the first intraperitoneal injection. Theblood and pancreatic tissue were harvested. General observation of morphology of thepancreas, the histopathology score of pancreatic, the levels of malondialdehyde and the activity of lipase, trypsin and SOD were determined. The data were analyzed by SPSS16.0.3. The molecular mechanisms of the therapeutic effects of AS on AP⑴The isolation and culture of pancreatic acinar cells from ratThe primary pancreatic acinar cells were isolated by collagenase digestion of SD rats.The primary pancreatic acinar cells were divided into normal group, LPS group, AS+LPSgroup, AS group. The final concentration of LPS was1μg/mL. The AS+LPS group waspretreated with10μg/mL AS, and then LPS was added after2hours. The cells andsupernatants were harvested6hours later.⑵After rat pancreatic acinar cells were stimulated with LPS, the mRNA and proteinexpression of TNF-α, IL-1β and IL-6was determined by semi-quantitative RT-PCR andELISA, respectively.⑶Semi-quantitative RT-PCR method was used to detect the effects of AS on ratpancreatic acinar cells TLR2, TLR4, and nuclear factor κB (NF-κB) p65mRNA expression.Results：1.The general observation of pancreatic tissue: the pancreas of AP induction groupwere significantly edema and darker than the control group. The texture of pancreas APinduction group turned hardens and surround by a little of bleeding spots. Compare to theAP induction group, the pancreas of AS treatment group was slightly edema, less bleedingspots and the color of pancreas was close to the control group. The pancreas coefficientwere significantly decreased, but no significant difference among the sub-groups of AStreatment group.2.Morphologic changes. The control group showed that the pancreatic cells arranged inneat rows, the duct of pancreatic intralobular was natural and the structure was neat.Pancreas lobular disordered arrangement, limitations interlobular distance widened,interlobular visible a few inflammatory cells and red blood cells were observed in APinduction group. The AS treatment group showed that slightly increased bleeding, lessnecrosis and interstitial a small amount of inflammatory cells.3.Pancreatic pathological score results. The scores of pathological changes of pancreasin treatment group were significantly lower than those of AP induction group (P<0.05).4. Pancreatic coefficient results: The pancreatic coefficient of AP induction group weresignificantly higher than those of control group (P<0.05). Compared with the AP induction group, the pancreatic coefficient f of AS treatment group was significantly decreased (P<0.05), but there is no significant difference among the sub-groups of AS treatment group.5. The biochemical parameters of pancreatic tissue and serum⑴Compared with the control group, the activity of serum amylase, lipase and trypsinof AP induction group was significantly increased (P <0.01); while compared with APinduction group, the activity of serum amylase, lipase and trypsin of AS treatment groupwas significantly decreased (P <0.05).⑵SOD and MDA results.①The activity of SOD in pancreatic homogenates in APinduction group was significantly lower than that in control group(P<0.01). After treated byAS, the activity of SOD was significantly higher than AP induction group (P<0.01).②Thelevels of MDA in pancreatic homogenates in AP induction group was significantly higherthan that in control group (P<0.01). After treated by AS, the activity of MDA wassignificantly lower than AP induction group (P<0.01).6. AS can decrease the secretion of IL-1β and IL-6which is induced by LPS in ratpancreatic acinar cells.7. AS can inhibit the expression of TLR2, TLR4and p65on LPS-induced acutepancreatitis rat pancreatic cellsConclusion：1. AS have a significant therapeutic effect on acute pancreatitis mouse model byinhibiting the activity of serum amylase, pancreatic lipase and proteases, and reducing thepancreatic tissue injury. After treated with AS, the activity of SOD was significantlyincreased while the level of MDA was significantly decreased.2. AS have a significant therapeutic effect on severe acute pancreatitis animal model byinhibiting the activity of serum AMS and lipase and reducing the pancreatic tissue lesions.After treated with AS, the activity of SOD was significantly increased while the level ofMDA was significantly decreased.3. The molecular mechanisms of the therapeutic effects of AS on acute pancreatitismay be related to inhibition of TLR2, TLR4and NF-κB expression, thus reducing therelease of pro-inflammatory cytokines such as IL-1β and IL-6.