| Da-Jian-Zhong decoction is first recorded in "The Synopsis of the Golden Chamber".It can warm the middle and nourish the deficiency,reduce the adverse effect and relieve pain,but it is currently less clinically used.The medical records published in the Chinese literature include gastritis,spleen-yang deficiency,pancreatic cancer,hydrocele,Intestinal obstruction,impotence,abdominal pain,etc.Da-Jian-Zhong decoction lacks highly effective indications and adapted patients,and its mechanism of action and the basis of medicinal substances are not clear,which limits the clinical application of Da-Jian-Zhong decoction and the development of its preparations.Objective:The clinical indications of Da-Jian-Zhong decoction are analyzed through the research of ancient books and literature,and the appropriate clinical application will be obtained,which lay a foundation for the subsequent research on the efficacy and mechanism.Then establish an animal model of spleen-yang deficiency syndrome to evaluate the efficacy of Da-Jian-Zhong decoction in improving the syndrome.Then transcriptome sequencing,q RT-PCR,Western Blotting and other methods are used to explore the mechanism of its efficacy.Finally,UPLC-Q Exactive-MS is used to identify the main components of Da-Jian-Zhong decoction,which is a preliminary study for the future study of its medicinal substances,and then lays the research foundation for the development of Da-Jian-Zhong decoction preparations.Methods:1.Literature research: Relevant ancient books were used to systematically verify the efficacy and clinical application of the prescription,and then determine the appropriate clinical application of Da-Jian-Zhong decoction.2.Research on the efficacy of Da-Jian-Zhong decoction for warming and nourishing deficiency: Establish a rat model of spleen-yang deficiency syndrome,and systematically evaluate the efficacy of Da-Jian-Zhong decoction in improving spleen-yang deficiency syndrome through macroscopic indicators such as body weight,rectal temperature,food intake,exercise ability,as well as organ index and pathological observations.3.Research on the mechanism of Da-Jian-Zhong Decoction in improving spleen-yang deficiency syndrome: Transcriptome sequencing technology was used to systematically mine the differentially expressed genes before and after the model building and before and after treatment with Da-Jian-Zhong decoction,and explore the mechanism of improving the spleen-yang deficiency syndrome through the evaluation of biological information.At the same time,q RT-PCR was a method used to verify the results of RNA-seq,and western blotting verify the key pathway.4.UPLC-Q Exactive-MS was used to identify the main ingredients in Da-Jian-Zhong decoction,and lay the foundation for the next step of medicinal substances research.Results:1.By analyzing the ancient literature of Da-Jian-Zhong decoction,it was found that among the main symptoms of Da-Jian-Zhong decoction,"various deficiency syndromes" are the most frequent,followed by "qi inverse" and "cold and pain in the heart and chest".Zanthoxylum bungeanum and dry ginger are hot,belonging to the spleen,stomach,and kidney.Yitang warms and nourishes the deficiency,ginseng benefits the spleen and qi.Therefore,our research is mainly to study the mechanism of the prescription for warming and nourishing the yang.2.Da-Jian-Zhong decoction significantly improved the symptoms of decreased body weight,rectal temperature,food intake,exercise capacity,and organ index in rats with spleen-yang deficiency syndrome;and effectively improved the pathological changes of the stomach,ileum,and colon of the digestive tract.The difference in the improvement effect of each group suggests that the middle and low-dose groups of Da-Jian-Zhong decoction can improve the pathological changes of spleen-yang deficiency syndrome,and the high-dose group and the positive group have the best improvement effect.3.Through transcriptome sequencing,the significantly differentially expressed genes were screened to obtain the gene set C＿vs＿M＿vs＿T,and this gene set was analyzed by bioinformatics.Through protein-protein interaction analysis,the 20 most important genes were screened: Cxcr5,Bcl6,Cxcr7,Il21 r,Gpr174,Sell,Cd79 a,Cxcr4,Ikzfl,Cxcl13,Gzmb,Pou2 afl,Cd27,Btk,Ptgs2,Cd72,Ikzf3,Ccl9,Pdyn,Idol.Through GO annotation,significant enrichment of immune-related biological functions were obtained such as the B cell receptor signaling pathway,positive regulation of leukocyte activation,lymphocyte activation and regulation of immune response.Annotated by KEGG,enriched immune-related pathways such as B cell receptor signaling pathway,intestinal immune network,and NF-κB signaling pathway.And the reliability of the sequencing results was verified by q RT-PCR quantitative analysis.In addition,Western Blotting was used to verify and analyze the differential genes(PKCβ,BCR,BTK)expressed in the most critical pathway——B cell receptor signaling pathway,and the expression change trend was consistent with the sequencing results,suggesting that the B cell receptor signaling pathway may be the key mechanism of Da-Jian-Zhong decoction to improve spleen-yang deficiency syndrome.4.Through UPLC-Q Exactive-MS,a preliminary study on the main components of Da-Jian-Zhong decoction was conducted,and a total of 19 components were identified,mainly including sterols,phenols,alkaloids,and flavonoids.Conclusion:According to literature studies,"various deficiency syndromes" are the most commonly treated symptoms of Da-Jian-Zhong decoction.Therefore,this research uses a compound method to establish a rat model of spleen-yang deficiency syndrome,and evaluates its efficacy through macroscopic evaluation and HE staining pathology.It was found that Da-Jian-Zhong decoction can significantly improve the spleen-yang deficiency syndrome.And the key mechanism related to the regulation of immunity and especially B cell receptor signaling pathway.The 19 components identified by UPLC-Q Exactive-MS are the possible medicinal substances. |
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