Based On TRPA1 Channel To Explore The Mechanism Of Lizhong Wan, Xiaojianzhong Decoction And Wuzhuyu Decoction In The Treatment Of Chronic Pancreatitis

Background:Lizhong wan(LZW),Xiaojianzhong tang(XJZT)and Wuzhuyu tang(WZYT)are classic prescriptions in Treatise on Febrile Diseases.They are effective in dispersing cold and relieving pain.They are classified as interior-warming agents in the textbook of Chinese medicine prescription,and are mainly used for treating cold syndrome of middle-jiao.Epigastric pain is one of the main manifestations of middle-jiao deficiency cold syndrome,which can be effectively alleviated by the treatment of dispersing cold.As a member of the middle-jiao,the pancreas is often affected by inflammation and suffers from pain.Lizhong wan,Xiaojianzhong tang and Wuzhuyu tang(three prescriptions for warming interior)are all effective in treating cold pain.Some modem studies have proved that they are used to treat chronic pancreatitis by somg Chinese doctors.Thermosensitive channel TRPA1 plays an important role in the pathogenesis of the chronic pancreatitis.TRPA1 mediates neuronal inflammation and hyperalgesia of pancreas.Blocking TRPA1 can significantly relieve pain and inflammation of pancreatitis.It has reported that some components such as gingerol(from ginger in LZW,XJZT and WZYT),cinnamaldehyde(from cinnamon in XJZT),evodiamine(from evodia rutaecarpa in WZYT)can regulate TRPA1.The treatment on the chronic pancreatitis by the three prescriptions may be realized by activating,desensitizing or inhibiting of TRPA1 on relevant sensory nerves.In this study,the chronic pancreatitis model of mice were induced by long-term repeated intraperitoneal injection of cerulein,than were treated each by the 3 prescriptions.The pain behavior of mice,the myeloperoxidase(MPO)and serum amylase(Amy)in pancreas tissue of the mice were tested.Immunohistochemistry and Western blot for TRPV1,PAR2,cPLA2 and EP4 expression in pancreatic tissues in the model mice were detected to reveal the molecular biological mechanism of Lizhongwan,Xiaojianzhong tang and Wuzhuyu tang in treating the chronic pancreatitis.Objective:To explore the mechanism of TRPA1 in the treatment of chronic pancreatitis by Lizhongwan,Xiaojianzhong tang and Wuzhuyu tang,so as to provide experimental basis for clarifying the scientific connotation of the efficacy of the formula and guide the clinical practice of traditional Chinese medicine.Methods:1.In this study,the chronic pancreatitis model of mice was induced by long-term repeated injections of cerulein.Lizhongwan,Xiaojianzhong tang and Wuzhuyu tang were used for intervention to observe the effects on pain behavior of mice.2.The model of chronic pancreatitis in mice was replicated.The blood of mice was taken for the detection of serum amylase,the pancreatic tissue of mice was taken for the detection of myeloperoxidase activity by ELISA,and HE staining was used for the pathological grading of pancreatic tissue to observe the histopathological changes.3.In the mouse chronic pancreatitis model,the expression of TRPA1,PAR2,cPLA2 and EP4 receptors in mouse pancreatic tissue was detected by immunohistochemistry and Western Blot,and the expression of TRPA1,PAR2,cPLA2 and EP4 in mouse DRG was detected by immunofluorescence.Result:1.Behavioral experiment of chronic pancreatitis in mice.In the mechanical pain experiment,after modeling by intraperitoneal injection of cerulein,the sensitivity of the model group to fiber filaments was enhanced,and the difference was statistically significant(P<0.01).After treatment,the sensitivity of Lizhong wan group,Xiaojianzhong tang group and Wuzhuyu tang group was decreased compared with the model group,and the difference was statistically significant(P<0.01).2.Physiological and pathological experiments of mouse CP.The degree of pancreatic injury in the model group is obvious.Compared with the normal group,the pathological score is higher,and there is statistical significance between the two(P<0.01).Compared with Lizhong wan,Xiaojianzhong tang and Wuzhuyu tang,the pathological score of the three groups is lower than that of the model group after the three-party treatment,and there is statistical significance between the two(P<0.01).Compared with the normal group,the MPO of mice was significantly increased after cerulein was established.The difference between the two groups was statistically significant(P<0.01).After drug administration,the MPO values of mice in the positive control group and the traditional Chinese medicine treatment group were lower than those in the model group.the difference between the groups and the model group was statistically significant(P<0.01).The AMY value in the blood of mice in the model group was higher than that in the normal group,and the difference was statistically significant(P<0.01).Compared with the model group,the AMY value in the positive drug group,Lizhong wan group,Xiaojianzhong tang group and Wuzhuyu tang group was significantly lower,and the difference was statistically significant(P<0.01).3.Immunohistochemistry and Western Blot of Pancreas in CP MiceEffect of Lizhong wan,Xiaojianzhong tang and Wuzhuyu tang on expression of TRPA1 in pancreatic tissue of mice.The expression of TRPA1 in pancreatic tissue of the model group was higher than that in the normal group(P<0.01).After administration of traditional Chinese medicine,compared with that in the model group,the expression of TRPA1 decreased and the difference was statistically significant(P<0.01).Effect of three parties on expression of PAR2 in pancreatic tissue of mice.After modeling,the expression of PAR2 in the pancreatic tissue of mice was higher than that of the normal group,and the difference was statistically significant(P<0.01).Compared with the model group,the expression of PAR2 in the pancreatic tissue of mice was significantly decreased after administration of Lizhong wan group,Xiaojianzhong tang group and Wuzhuyu tang group,and the difference was statistically significant(P<0.01).Effect of three parties on expression of cPLA2 in pancreatic tissue of mice.After modeling,the expression of cPLA2 increased compared with the normal group,and the difference was statistically significant(P<0.01).after three-way administration,the expression of cPLA2 in pancreatic tissue decreased compared with the model group,and the difference was statistically significant(P<0.01).Effect of three parties on expression of EP4 in pancreatic tissue of mice.The content of EP4 in pancreatic tissue was significantly increased after cerulein modeling,and the difference was statistically significant(P<0.01).after drug administration,the content of E P4 in treatment group was significantly decreased,and the difference was statistically significant(P<0.01).Conclusion:Lizhong wan,Xiaojianzhong tang and Wuzhuyu tang can reduce the expression of PAR2,cPLA2,EP4 and TRPA1 receptor in pancreatic tissue,inhibit the activity of TRPA1 channel,reduce the release of downstream inflammatory substances,improve pancreatitis and relieve abdominal pain.