Exploring The Molecular Mechanism Of Zuojin Pill In Treating Gastroesophageal Reflux Disease Based On Bitter Taste Receptors And Capsaicin Receptors

Background and PurposeZuojin Pill,originating from the book "Danxi Xin fa" written by Zhu Danxi,is a representative formula combining cold and heat.Multiple clinical consensus opinions and textbooks recommend Zuojin Pill for the treatment of gastroesophageal reflux disease(GERD).Through preliminary research and literature review,the research team found a close relationship between Zuojin Pill treatment for GERD and bitter taste receptors(TAS2Rs)and transient receptor potential vanilloid 1(TRPV1)which also known as capsaicin receptors.TAS2Rs and TRPV1 are widely expressed in the digestive tract and have synergistic effects on biological effects such as esophageal lower esophageal sphincter contraction,gastric emptying,and visceral sensation.Biological synergy refers to the interaction between targets,which may be an important entry point for studying the efficacy of traditional Chinese medicine formulas.Therefore,this study aims to elucidate the molecular mechanisms and scientific implications of Zuojin Pill in the treatment of GERD from a new perspective based on bitter taste receptors and capsaicin receptors.Research Contents and ResultsChapter one of this study,we constructed a rat model of GERD using esophagogastric anastomosis.After four weeks,rats were orally administered Zuojin Pill at doses of 0.63,1.26,and 2.52 g/kg for four consecutive weeks.The results showed that Zuojin Pill could inhibit the papillary hyperplasia and basal layer thickening of esophageal tissues in GERD rats,as well as suppress the expression of inflammatory factors IL-1β,IL-6,and TNF-α.Immunofluorescence,immunohistochemistry,Western blot,and transmission electron microscopy techniques confirmed that Zuojin Pill could improve the barrier function and ultrastructure of esophageal tissues in GERD rats.Utilizing bioinformatics techniques,CIBERSORTX,and immunofluorescence,it was demonstrated that Zuojin Pill could upregulate CD206 expression and downregulate iNOS and CD86 expression,promoting macrophage polarization towards the M2 phenotype.To further elucidate the potential mechanisms of Zuojin Pill,bioinformatics techniques were employed to predict their association with the MAPK and NF-κB signaling pathways in GERD treatment.Western blot and immunofluorescence techniques confirmed that Zuojin Pill could inhibit the phosphorylation of proteins related to the MAPK and NF-κB signaling pathways.Chapter two of this study,we utilized an acidic culture medium combined with mixed bile acid salts to treat three types of normal esophageal epithelial cells to screen for the optimal modeling conditions and cells.Using this as a model,cells were treated with 25,50,and 100 μM berberine and 1,5,and 10 μM evodiamine.The results showed that both berberine and evodiamine could inhibit the expression of inflammatory factors in the GERD cell model and upregulate the expression of tight junction-related proteins.Their mechanisms of action may be related to the inhibition of the MAPK and NF-κB signaling pathways.Molecular docking and molecular dynamics simulation analyzed the binding modes of berberine with TAS2R38 and evodiamine with TRPV1,as well as the stability of the binding conformations.The use of bitter taste receptor inhibitors and capsaicin receptor inhibitors confirmed that berberine activated bitter taste receptors and evodiamine activated thermosensitive receptors,promoting intracellular Ca2+influx.Transfection with TAS2R38-siRNA and TRPVl-siRNA could respectively inhibit the effects of berberine and evodiamine on intracellular Ca2+influx,inflammatory factors,tight junction-related proteins,as well as the phosphorylation of proteins related to the MAPK and NF-κB signaling pathways.Using multiple synergy evaluation calculation models,it was found that there was a strong synergistic effect between berberine and evodiamine.Chapter three of this study,we aimed to establish a rat model of GERD to explore whether berberine and evodiamine are the key effective components of Zuojin Pill in treating GERD.The results showed that both berberine and evodiamine,when administered alone or in combination,could inhibit inflammatory factors,enhance barrier function,regulate macrophage polarization,and suppress the MAPK and NF-κB signaling pathways.Using the above-mentioned detection indicators,bitter taste receptor inhibition and TRPV1 chemical silencing methods were employed to evaluate the efficacy of berberine and evodiamine in treating GERD.The results showed that inhibition of bitter taste receptors and chemical silencing of TRPV1 could significantly attenuate the therapeutic effects of berberine and evodiamine in treating GERD.Conclusion and SignificanceZuojin Pill exert their therapeutic effects on GERD by inhibiting the MAPK and NF-κB signaling pathways,thereby suppressing inflammatory factors,enhancing esophageal barrier function,and regulating macrophage polarization.Berberine and evodiamine are the key effective components of Zuojin Pill in treating GERD,as they can respectively activate TAS2R38 and TRPV1,leading to the inhibition of inflammation,upregulation of barrier function,and suppression of the MAPK and NF-κB signaling pathways.This study found that TAS2R38 and TRPV1 could serve as potential targets for GERD treatment,revealing the specific molecular mechanisms underlying the treatment of GERD by Zuojin Pill and its key effective components berberine and evodiamine.This provides important research evidence for the use of traditional Chinese medicine in treating GERD and offers scientific insights into the compatibility of Coptidis Rhizoma and Evodiae Fructus in Zuojin Pill.