Dachengqi Decoction And Emodin In The Treatment Of Severe Acute Pancreatitis By Inhibiting NADPH Oxidase Activity

Objective:By replicating the rat and mouse severe acute pancreatitis(SAP)model,the effects of Dachengqi decoction,the classic prescription of Purgative therapy and its main component emodin on NADPH oxidase were investigated.Besides,the oxidative stress and inflammasomes are also studied for their mechanism of action against SAP.Methods:1.The inhibitory effect of Dachengqi Decoction on NADPH oxidase in SAP rats: 24 SD rats were retrogradely injected with 5% sodium taurocholate to replicate the SAP rat model.They were randomized into 3 groups with 8 rats in each and named sham operation group,SAP group and SAP+Dachengqi decoction group(also known as Dachengqi group)respectively.Rats in each group were executed 6 hours after modeling,and the caudal ventral midline coeliotomy then was performed to collect blood sample from abdominal aorta and take the pancreatic tissue.The levels of Amy,IL-1,IL-1? and TNF-? in the supernatant of blood samples were detected by ELISA,and the levels of oxidative stress indicators SOD,MDA and GSH-PX in pancreatic tissue homogenate also observed in the same way.HE staining was performed after sectioning of pancreatic tissue so as to do pathological scoring.Western blot and immunohistochemistry were used to detect the expression levels of NLRP3,NADPH oxidase 2(NOX2)and NOX main structural subtypes P22 and P47 in pancreatic tissue.2.Experimental study of emodin in the treatment of SAP mice by inhibiting NADPH oxidase: Based on the model of SAP mice induced by cerulein+LPS,five groups with 8 mice in each which were named the control group,SAP group,SAP + Dachengqi decoction group(also known as Dachengqi decoction group),SAP + emodin group(also known as emodin group)and SAP + DPI group(DPI,a kind of NADPH oxidase inhibitor)were set up,Mice in each group were executed 12 hours after modeling to collect blood from the eyelids and also take the pancreatic tissue.The levels of Amy,IL-1,IL-1? and TNF-? in serum were detected by ELISA kit.The levels of oxidative stress indicators SOD,MDA and GSH-PX in pancreatic tissue homogenate were detected by kit.HE staining and pathological scoring were performed after sectioning of pancreatic tissue.Western blot and immunohistochemistry were used to detect the expression level of NLRP3,NOX2,P22 and P47 in pancreatic tissue.Results1.The inhibitory effect of Dachengqi Decoction on NADPH oxidase in SAP rats: Compared with the sham operation group,the serum levels of Amy,IL-1,IL-1? and TNF-? in the SAP group were significantly increased;Among the indicators in oxdative stress in pancreas,the level of MDA in SAP group was significantly increased,while the levels of SOD and GSH-PX were significantly decreased.Dachengqi decoction could significantly reduce the increased MDA in SAP group and increase the level of SOD and GSH-PX in SAP group(P <0.05).HE results suggest that the pancreatic tissue of SAP group has obvious hemorrhage,necrosis and inflammatory cell infiltration compared with sham operation group.Dachengqi decoction can significantly improve the pathological changes of pancreatic tissue and reduce the pathological score of SAP pancreas(P<0.05).The expression level of related protein in pancreatic tissue detected by Western blot suggested that protein level in NLRP3,NOX2,P22 and P47 was significantly higher than that in sham operation group(P<0.05).Dachengqi decoction could significantly downregulate these indexes in SAP.(P<0.05),immunohistochemistry results also detected the increase of the positive expression level of target protein in SAP group and inhibiting effect Dachengqi decoction has to this increase(P<0.05).2.Experimental study of emodin in the treatment of SAP mice by inhibiting NADPH oxidase: Compared with the control group,the values of serum indexes of Amy,IL-1,IL-1? and TNF-? as well as the oxidative stress index of pancreatic tissue MDA were significantly increased while the values of SOD and GSH-PX decreased,and the levels of Amy,IL-1,IL-1?,TNF-? and MDA in Dachengqi Decoction group,emodin group and DPI group were lower than those in SAP group(P<0.05).The levels of SOD and GSHPX were significantly increased(P<0.05).The results of HE showed that the pancreatic tissue of SAP group had obvious inflammation and necrotic changes compared with the control group.The pathological changes of pancreas in Dachengqi decoction group,emodin group and DPI group were improved than that in SAP group,which manifested as lower pathological score(P<0.05).The protein level in NLRP3,NOX2,P22 and P47 in pancreatic tissue of mice,which were detected by Western blot suggested that the protein level in SAP group was significantly higher than that in the control group(P<0.05).While the level decreased in Dachengqi decoction group,emodin group and SAP+DPI group(P<0.05);Dachengqi decoction group,emodin group and DPI group had little difference in protein expression level(P>0.05);Immunohistochemistry experiments also further confirmed changes in protein expression levels which was consistent with trends in western blot results.Conclusion:1.Dachengqi Decoction can reduce the expression of NADPH oxidase and the level of oxidative stress in SAP rat pancreas,reduce inflammatory factors in SAP rats as well as pancreatic damage.2.Dachengqi Decoction and its main component,emodin,can improve SAP by inhibiting the expression of NADPH enzyme and reducing the expression level of inflammatory factors and inflammatory NLRP3.