| Background:Xiaojianzhong tang,Wuzhuyu tang and Lizhong wan are the famous formulas in shanghanlun.They have the effect of dispelling cold and relieving pain and often be used in the treatment of middle-focus abdominal pain disease.The pancreas belongs to the middle focus category.Non-infectious inflammation usually occur in pancreas by various causes and exhibit pain.Modern studies have shown that thermosensitive channels distributed on sensory nerve nociceptors can mediate a variety of acute and chronic inflammation and pain,thermosensitive channel TRPV1 plays a key role in the pathogenesis of acute pancreatitis and pancreatic pain.Theoretically,blocking this pathway to reduce the perception and transmission of noxious sensation is an effective way to eliminate inflammation and relieve pain.Previous studies have shown that the three formulas contain abundant thermosensitive channel TRPV1 agonists/inhibitors.They may play a role in alleviating pancreatic inflammation and relieving pain by activating,desensitizing/inhibiting the thermosensitive channel TRPV1.In this study,a mouse model of acute pancreatitis was induced by cerulean.The levels of serum amylase,myeloperoxidase,histopathology,animal pain behaviour,immunohistochemistry and Western blot were measured to explore the molecular biological mechanism of the three prescriptions in the treatment of acute pancreatitis.Objective:To explore the mechanism of Xiaojianzhong tang,Wuzhuyu tang and Lizhong wan on reducing inflammation and relieving pain in acute pancreatitis,and provide scientific basis for clinical rational drug use and further development.Methods:Thirty male mice of SPF grade(C57BL/6)weighing 20+2g were randomly divided into five groups:normal group,model group,Xiaojianzhong tang group,Wuzhuyu tang group and Lizhong wan group,with 6 mice in each group.The mice in the model group and each treatment group were given intraperitoneal injection of cerulein(50?g/kg,100?L/mice/time),repeated every 1 hour for 8 times.Each group was given three times,the first time 1 hour after the first intraperitoneal injection,the second time 1 hour after the eighth injection and the third time24 hours after the first intraperitoneal injection.Xiaojianzhong granule(5.85g/kg·d),Wuzhuyu tang fine granule(0.78g/kg·d)and Lizhong wan(2.34g/kg·d)were administered to the three groups respectively.The pain behavioral response of mice was observed by hot plate test 1 hour before taking samples.Serum amylase level was detected by eyeball extraction after pentobarbital sodium anesthesia.Myeloperoxidase content was detected by ELISA and pathological changes of pancreas were observed by HE staining.The expression of TRPV1,NK1R(SP receptor)and CGRP in mouse pancreas was detected by immunohistochemistry and Western blot.Results:(1)Hot plate test in mice:Compared with the normal group,the first time of pain behavior in the model group was significantly shorter(P<0.05).Compared with the model group,the first time of pain response in mice was delayed in all groups(P<0.01 or P<0.05).(2)Biochemical changes of acute pancreatitis in mice:Compared with the normal group,the serum AMY level in the model group increased significantly(P<0.001).Compared with the model group,the serum AMY level in the treatment group was decreased(P<0.01 or P<0.001).Compared with the normal group,the content of MPO in pancreas tissue of model group mice increased(P<0.01).Compared with the model group,the MPO content of mice in each administration group decreased(P<0.05 or P<0.01).(3)Histopathological changes of acute pancreatitis in mice,HE staining showed that:In normal group,pancreatic acinar cells were intact,interlobular septa were normal,and no inflammatory cells were found.Compared with the normal group,after injection of cerulein,the pancreatic tissue of mice showed edema of acinar cells,serious vacuolation,enlargement of intercellular and lobular spaces,and infiltration of large numbers of red blood cells and inflammatory cells around blood vessels.Compared with the model group,after administration of Xiaojianzhong tang,Wuzhuyu tang and Lizhong wan,the septum of pancreatic lobules decreased,the degree of acinar cell edema and inflammatory cell infiltration decreased significantly.(4)Changes of related indicators of TRPV1 pathway:Immunohistochemistry showed that:Compared with the normal group,the expression of TRPV1 in pancreas tissue of model group was significantly increased(P<0.001);compared with model group,the expression of TRPV1 in pancreas tissue of mice treated with Xiaojianzhong tang,Wuzhuyu tang and Lizhong wan was significantly decreased(P<0.001).Compared with the normal group,the expression of NK1R(SP receptor)in pancreas tissue of mice in model group was significantly up-regulated(P<0.001);the expression of NK1R in pancreas tissue of mice treated with Xiaojianzhong tang,Wuzhuyu tang and Lizhong wan was significantly down-regulated(P<0.001).Compared with the normal group,the expression of CGRP in the pancreas tissue of the model group was significantly increased(P<0.001);compared with the model group,the expression of CGRP in the pancreas tissue of the mice in each treatment group was significantly decreased(P<0.001).Western Blot results show that:Compared with the normal group,the expression of TRPV1 protein in the pancreas of mice in the model group was significantly increased(P<0.001);the expression of TRPV1 in the pancreas of mice in each treatment group was significantly decreased after administration(P<0.001).Compared with the normal group,the expression of NK1R protein in pancreas of the model group was significantly increased(P<0.001);compared with the model group,the expression of NK1R protein in pancreas of mice in each treatment group was significantly decreased(P<0.001).Compared with the normal group,the expression of CGRP in pancreas of model group mice was significantly increased(P<0.001);compared with model group,the expression of CGRP in pancreas of mice in each treatment group was significantly decreased(P<0.001).Conclusion and significance:Xiaojianzhong tang,Wuzhuyu tang and Lizhong wan can alleviate the acute pancreatic inflammation induced by cerulein and delay the occurrence of pancreatic pain in mice.This effect may be achieved by reducing the release of SP and CGRP by down-regulating TRPV1(desensitization or inhibition of TRPV1 channel)in pancreas. |
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