To Investigate The Effect Of Wenzhong Quhan Recipe On Neurogenic Inflammation In Mice With Chronic Pancreatitis Based On TRPV1

Background:Wenzhong Quhan Fang Wuzhuyu Tang and Xiaojianzhong Tang are derived from Shanghanlun,and both have the effects of dispelling cold and relieving pain.They are widely used clinically for chest and abdomen diseases with pain as the main manifestation,especially digestive system diseases,such as chronic pancreatitis(CP),chronic gastritis,peptic ulcer,etc.The main pathological features of chronic pancreatitis are persistent inflammation,interstitial fibrosis and pancreatic gland destruction.The clinical manifestation is mainly recurrent chronic pain,causing serious damage to the quality of life of patients.Recent studies have shown that TRPV1 is a new target for regulating CP pancreatic neurogenic inflammation and chronic pain.Activation of TRPV1 induces pancreatic sensory nerve endings to release SP(by binding with NK1R to transduce pain signals)and CGRP to induce CP neurogenic inflammation and chronic pain.NGF strongly sensitizes TRPV1,increases TRPV1 transport,and amplifies pain.The components of Wuzhuyu Tang and Xiaojianzhong Tang contain a variety of compounds that can regulate TRPV1,such as reported[6,8,10]-gingerols,[6,8,10]-shogaols,evodiamine,rutaecarpine and cinnamaldehyde,which can regulate the activity of TRPV1 directly or indirectly.Therefore,we proposed the hypothesis that Wuzhuyu Tang and Xiaojianzhong Tang could affect the release of downstream SP and CGRP by regulating the activity of TRPV1,and alleviate the neurogenic inflammation and chronic pain of CP.In this study,CP mouse model was induced by caerulein,and the behavioral,histopathological and molecular biological methods were used to study the mechanism of Wuzhuyu Tang and Xiaojianzhong Tang in the treatment of CP by taking NGF-TRPV1-NK1R(SP)/CGRP axis as the object of investigation,so as to enrich the prescription connotation of "warming method" from the molecular level,and provide experimental support for the precise use of clinically effective prescriptions of traditional Chinese medicine.Objective:Taking NGF-TRPV1-NK1R(SP)/CGRP axis as the investigation object,this study explored the effects and molecular mechanism of Wuzhuyu Tang and Xiaojianzhong Tang on CP neurogenic inflammation and chronic pain,providing experimental basis for clarifying the scientific connotation of "warming method" of the prescription and providing ideas for TCM treatment of CP.Methods:Study one:Hot plate test and HE staining were used to observe the effects of Wuzhuyu Tang and Xiaojianzhong Tang on the pain behavior and histopathology of CP model mice induced by caerulein.Study two:Immunohistochemical method was used to detect the protein expression of inflammatory factors IL-1β,IL-6,TNF-α and NGF in pancreatic tissue of CP model mice.Western blot was used to detect the protein expression of TRPV1,NK1R and CGRP in the pancreatic tissue of CP model mice,to further explore the mechanism of Wuzhuyu Tang and Xiaojianzhong Tang on pancreatic inflammation and pain behavior of CP model mice.Results:(1)Hot plate test:Compared with the normal group,the time required for the first appearance of pain behavior in mice in the model group was significantly shorter(P<0.01).Compared with the model group,the time required for the first appearance of pain behavior in mice in Wuzhuyu Tang group and Xiaojianzhong Tang group both prolonged(P<0.05).(2)Histopathological changes:HE staining results showed that compared with the normal group,the pancreatic tissue of the model group had obvious acinar cell atrophy,cell gap expansion,lobule structure destruction and interstitial inflammatory cell infiltration.Wuzhuyu Tang group and Xiaojianzhong Tang group compared with the model group,acinar cell atrophy was significantly reduced,pancreatic tissue structure was significantly restored,interstitial inflammatory cell infiltration was significantly improved.(3)Changes of inflammatory factors in pancreatic tissue:Immunohistochemical results showed that compared with the normal group,the expression of IL-1β protein in the model group increased(P<0.05);compared with the model group,the expression of IL-1β protein in the Wuzhuyu Tang group and the Xiaojianzhong Tang group had no significant change(P>0.05).Compared with the normal group,the IL-6 protein expression in the model group increased(P<0.01);compared with the model group,the IL-6 protein expression in the Wuzhuyu Tang group had no significant change(P>0.05),and the IL-6 protein expression in the Xiaojianzhong Tang group decreased(P<0.05).Compared with the normal group,the expression of TNF-αprotein in the model group increased significantly(P<0.001);compared with the model group,the expression of TNF-α protein in the Wuzhuyu Tang group decreased(P<0.05),and the TNFa protein expression in the Xiaojianzhong Tang group significantly decreased(P<0.01).(4)Changes of NGF-TRPV1-NK1R(SP)/CGRP pathway proteins:Immunohistochemical results showed that compared with the normal group,the expression of NGF protein in pancreatic tissue of mice in the model group significantly increased(P<0.01);compared with the model group,the expression of NGF protein in pancreatic tissue of mice in Wuzhuyu Tang group and Xiaojianzhong Tang group decreased(P<0.01).Western blot results showed that compared with the normal group,the expression of TRPV1 protein in pancreatic tissue of mice in the model group increased(P<0.01).Compared with the model group,the expression of TRPV1 protein in pancreatic tissue of mice in Wuzhuyu Tang group and Xiaojianzhong Tang group decreased(P<0.05).Compared with the normal group,the expression of NK1R protein in pancreatic tissue of mice in the model group increased(P<0.05).Compared with the model group,the expression of NK1R protein in pancreatic tissue of mice in Wuzhuyu Tang group and Xiaojianzhong Tang group decreased(P<0.05).Compared with normal group,the expression of CGRP protein in pancreatic tissue of mice in the model group increased(P<0.01).Compared with the model group,the expression of CGRP protein in pancreatic tissue of mice in Wuzhuyu Tang group and Xiaojianzhong Tang group decreased(P<0.05).Conclusion:(1)Wuzhuyu Tang and Xiaojianzhong Tang could relieve the pancreatic inflammation and pain in CP model mice.(2)The anti-inflammatory and analgesic effects of Wuzhuyu Tang and Xiaojianzhong Tang on pancreatitis in CP model mice are related to the inhibition of the protein expression of NGF-TRPV1-NK1R(SP)/CGRP axis associated with neuritis and pain.