The Material Basis And Mechanism Investigation Of The Gentianae Macrophyllae Radix-Chuanxiong Rhizoma Drug Pair For Treating Ischemic Bowel Disease Based On The Principle Of “Xuanbi Tongluo”

BackgroundIschemic bowel disease(ICBD),manifested in local intestinal tissue necrosis because of a lack of sufficient intestinal blood supply,is mainly caused by mesenteric ischemia.This kind of disease is characterized by high mortality rate and complex pathological mechanism,and there is no ideal treatment at present.Traditional Chinese Medicine has significant therapeutic effects in preventing and treating common and frequently-occurring diseases.ICBD belongs to the “Changbi” syndrome,with the main pathogenesis of qi stagnation and blood stasis,arthralgia and obstruction,and collateral qi stagnation,which,therefore,can be treated based on the principle of “Xuanbi Tongluo”.Gentianae Macrophyllae Radix and Chuanxiong Rhizoma are the main ingredients in famous prescriptions for treating ischemic diseases.such as Daqin Geng Decoction,Juanbi Decoction,and Shentong Zhuyu Decoction,suggesting that Gentianae Macrophyllae Radix and Chuanxiong Rhizoma play an important role in the treatment of ICBD.Exploring the compatibility rules of Gentianae Macrophyllae Radix and Chuanxiong Rhizoma in prescriptions and clarifying the material basis and mechanism of their efficacy can provide new ideas for the clinical treatment of ICBD.Aims1.Exploration of the rules of drug proportioning: Explore the medication rules of prescriptions containing Gentianae Macrophyllae Radix and Chuanxiong Rhizoma,clarify the effects of different proportions of Gentianae Macrophyllae Radix and Chuanxiong Rhizoma on ICBD,and determine the optimal proportion of Gentianae Macrophyllae Radix and Chuanxiong Rhizoma for treating ICBD.2.Confirmation of the pharmacodynamic material basis: Identify the effective ingredient group based on the optimal proportion of Gentianae Macrophyllae Radix and Chuanxiong Rhizoma to exert their pharmacodynamic effects,and confirm the efficacy of the effective ingredient group in treating ICBD.3.Clarification of the mechanism of drug efficacy: Clarify the efficacy mechanism of the effective ingredient group in treating ICBD through its effects on intestinal vascular permeability,intestinal mucosal integrity,and intestinal adhesion.Methods1.Exploration of the rule of drug proportioningA mouse disease model was established by clamping the superior mesenteric artery,and the animals were orally administered with different ratios of Gentianae Macrophyllae Radix and Chuanxiong Rhizoma extracts by gavage.The intestinal mucosal injury and intestinal barrier function were detected by HE staining,ELISA i FABP,D2 D,AB-PAS staining,and immunofluorescence methods after drug intervention,and the intestinal vascular permeability after injury was detected by Evans blue staining.The intestinal propulsion experiment was performed to detect the peristaltic function of the small intestine in mice.Sirius red staining was used to detect the collagen deposition in the site of intestinal adhesion.The effects of different proportions of Gentianae Macrophyllae Radix and Chuanxiong Rhizoma on ICBD were clarified through the above methods,and the optimal drug proportion for treating ICBD was determined.2.Confirmation of the pharmacodynamic material basisBased on the key pathological processes of ICBD,we respectively established a disease target library and a target library associated with the Gentianae Macrophyllae Radix and Chuanxiong Rhizoma drug pair by employing genecard and pharmmapper databases.The intersection of the two libraries is the potential target for the treatment of ICBD with the drug pair.We analyzed and identified the differential components that enter the blood and intestines after treatment with the Gentianae Macrophyllae Radix-Chuanxiong Rhizoma by means of UPLC-Q-TOF-MS/MS.We utilized Cytoscape software to analyze the correlation between the potential targets for the drug treatment of diseases and the identified components that enter the blood and intestine,and screened out the effective component groups for the treatment of ICBD with the drug pair.By measuring the standard curves of each component in the effective component group,we clarified the content and proportion of each component in the drug pair,and conducted experiments on efficacy in vivo accordingly,with the purpose of confirming the efficacy of the effective component group in the treatment of ICBD.3.Clarification of the mechanism of drug efficacyThe key pathological processes involved in the treatment of ICBD with the Gentianae Macrophyllae Radix-Chuanxiong Rhizoma are potentially related to the effects on vascular permeability and intestinal barrier function,as well as the formation of intestinal adhesions.Therefore,we selected target cells highly related to the pathological processes,including vascular endothelial cells,intestinal epithelial cells,and fibroblasts,and key pathways to investigate the mechanism of drug efficacy.We established OGD/R models of HUVEC and HIEC-6 cells,TGF-β-induced fibrosis models of L929 cells,and then respectively treated them with FAK pathway inhibitor Y15,MCLK pathway inhibitor ML-7,and SMAD3 pathway inhibitor SB431542 to explore the changes in key proteins of vascular permeability pathway FAK/VE-cadherin,intestinal barrier function pathway MLCK/MLC,and intestinal adhesion pathway TGF-β1/SMAD3 by immunofluorescence and western blotting methods.We finally clarified the efficacy mechanism of the functional ingredient group in the treatment of ICBD.Results1.Study on the relationship between different proportions and efficacy of Gentianae Macrophyllae Radix-Chuanxiong RhizomaThe results of data mining and association analysis showed that there were a total of 252 prescriptions in the Chinese Medicine Formula Dictionary that contained both Gentianae Macrophyllae Radix and Chuanxiong Rhizoma,with the most being those with a ratio of 1 between Gentianae Macrophyllae Radix and Chuanxiong Rhizoma.The treatment focus of different ratios of Gentianae Macrophyllae Radix-Chuanxiong Rhizoma varied,with the efficacy focusing on “Xuanbi” when the ratio was greater than 1,and on “Tongluo”when the ratio was less than 1.Based on the results of data mining,extracts were respectively prepared from the Gentianae Macrophyllae Radix alone,Chuanxiong Rhizoma alone,Gentianae Macrophyllae Radix and Chuanxiong Rhizoma at ratios of 1:1,1:2 and 2:1.In vivo experiments confirmed that different ratios of Gentianae Macrophyllae Radix-Chuanxiong Rhizoma could reduce vascular permeability,alleviate intestinal adhesion,improve intestinal epithelial tight junction,and thereby alleviate ICBD.After conducting a one-way analysis of variance on the efficacy of each group,we found that the efficacy of the drug pair was superior to that of the group alone,with the optimal efficacy achieved at a ratio of 1:2 between Gentianae Macrophyllae Radix and Chuanxiong Rhizoma.2.Study on the effect of Gentianae Macrophyllae Radix-Chuanxiong Rhizoma in the treatment of ischemic bowel diseaseThe drug pair was composed of Gentianae Macrophyllae Radix and Chuanxiong Rhizoma at a ratio of 1:2,which had the best therapeutic effect.The LD50 of the drug pair for oral administration was determined by the Horn method to be 75.5 g/kg of that of the crude drug.Based on the guiding principles of drug toxicology,preclinical safety evaluation and practice of new drugs,the low,medium and high doses of drugs for animal administration were determined to be 2 g/kg,4 g/kg and 8 g/kg,respectively.In vivo experiments confirmed that the drug pair of Gentianae Macrophyllae Radix and Chuanxiong Rhizoma could dose-dependently alleviate intestinal mucosal injury,reduce vascular permeability,alleviate intestinal adhesion,improve intestinal epithelial tight junction,maintain intestinal barrier function,and thus exert the efficacy in the treatment of ICBD.3.Excavation and confirmation of the efficacy of the main functional components in the drug pair for the treatment of ischemic bowel diseaseThrough UPLC-Q-TOF-MS/MS and drug-target interaction analysis,we identified the effective component group for the treatment of ICBD as ferulic acid,ligustilide,levistilide A,gentiopicrin,swertianin,sweroside,and loganic acid,totaling seven components.The content and molar ratio of each component were determined by measuring the standard curve of the effective component group,of which the molar ratio of the components was 36:2:1:190:4:6:40.The efficacy of the three-component group consisting of ferulic acid,gentiopicrin,and loganic acid,which had the highest content,was compared with that of the seven-component group.It was found that the efficacy of the seven-component group was significantly better than that of the three-component group,indicating that the remaining four also played a key role in the treatment of ICBD despite their low content.Therefore,the seven-component group was used as the key efficacy component group for the drug pair,and the efficacy of the effective ingredient group was confirmed through in vivo experimental systems.4.Clarification of the efficacy mechanism of effective ingredient group in the treatment of ischemic bowel diseaseBased on the key pathological processes of disease occurrence,we analyzed and identified potential pathways involving vascular endothelial cells,intestinal epithelial cells,and fibroblasts respectively as FAK/VE-cadherin,MLCK/MLC,and TGF-β1/SMAD3.Through in vivo and in vitro experiments,we confirmed that the effective ingredient group could reduce FAK phosphorylation levels,increase VE-cadherin expression,reduce vascular permeability,and protect HUVEC;the effective ingredient group could reduce MLCK expression,increase the expression of tight junction proteins ZO-1 and Occludin,and alleviate HIEC-6 injury;the effective ingredient group could reduce the phosphorylation level of SMAD3,reduce the expression of α-SMA and Collagen I,and inhibit the abnormal proliferation of L929 cells.The above results clarify the efficacy mechanism of the drug pair in the treatment of ICBD based on the theory of “Xuanbi Tongluo” through multiple components and pathways.ConclusionThis study is the first to discover that the drug pair of Gentianae Macrophyllae Radix-Chuanxiong Rhizoma at a ratio of 1:2 has the optimal treatment effect on ICBD.The effective ingredient group that exerts its efficacy includes ferulic acid,ligustilide,levistilide A,gentiopicrin,swertianin,sweroside,and loganic acid.It is also clarified that the effective ingredient group can reduce vascular permeability,maintain intestinal barrier function,and inhibit intestinal adhesion by regulating the FAK/VE-cadherin pathway based on vascular endothelial cells,the MLCK/MLC pathway in intestinal epithelial cells,and the TGF-β1/SMAD3 pathway in fibroblasts.This study provides not only new strategies and methods for the clinical treatment of ischemic bowel disease,but also a new model for the study of the material basis of Traditional Chinese Medicine.