Study On The Efficacy Of C0127 Combined With Low-dose Cyclosporine In The Prevention And Treatment Of Acute Graft-versus-host Disease In Mice And The Mechanism Of Intestinal Protection

Background:Allogeneic hematopoietic stem cell transplantation(allo-HSCT)is a mature and effective method for the treatment of malignant tumors and blood diseases.Acute graft-versus-host disease(aGVHD)is a common complication after transplantation,which seriously restricts the application and development of allo-HSCT and threatens the life safety of transplant patients.Graft-versus-host disease(GVHD)is driven by activated allogeneic donor T lymphocytes,which initiate cytotoxic attacks and cause damage to the skin,liver,and intestine of the recipient.Among them,the gastrointestinal tract plays a key role in GVHD.Gastrointestinal injury leads to systemic inflammation and lethal GVHD.As a routine clinical treatment,immunosuppressant cyclosporine injection(CsA)is often used as first-line prevention medicine,but at present,immunosuppressants are mostly aimed at single target,the prevention and treatment effect is not ideal,and most of them have side effects that damage the body.Due to the lack of specific treatment,the incidence rate and mortality rate of allo-HSCT recipients are still high.Traditional Chinese medicine is a unique excellent traditional culture in China,and herbal formulas have been used to treat human diseases for thousands of years.Xubijing injection(XBJ),composed of Carthami flos,Paeoniae radix rubra,Chuan-xiong rhizoma,Salviae miltiorrhizae radix,and Angelicae sinensis Radix,has the functions of promoting blood circulation and removing stasis,dispelling toxic pathogens,reducing inflammation and protecting organs.Our previous research found that the combination of XBJ and CsA can improve the survival of aGVHD mice,protect target organs and improve the disordered intestinal flora,and the main component composition C0127 in XBJ combined with CsA can also produce a prevention and treatment effect similar to XBJ,slow down the process of aGVHD,protect the target organs of mice and improve the disorder of intestinal flora.Based on the inevitable side effects of Western medicine,we tried to explore the efficacy of CsA combined with C0127 when the dose of CsA was reduced,so we made the following hypothesis:(1)C0127 combined with low-dose CsA can prevent aGVHD in mice;(2)The combination of C0127 and low-dose CsA has the effect of intestinal protection.Objective:Based on the previous research results,this project hopes to further explore whether C0127 can prevent and treat aGVHD mice under the condition of reducing the dose of CsA,achieve the effect of synergism and toxicity reduction,and further explore the protective effect of C0127 on the intestinal tract and its mechanism,so as to lay a preliminary foundation for clarifying the mechanism of XBJ.It provides a new way for the research and development of new Chinese medicine.Methods:(1)By referring to relevant literature on the composition of XBJ substances,the main pharmacodynamic components were found,and the main components were quantitatively analyzed by high-performance liquid chromatography(HPLC).C0127 was combined according to the concentration ratio in XBJ.(2)Mouse Acute graft-versus-host disease model was established,and the mice were randomly divided into 4 groups: Control group,aGVHD group,5 mg/kg CsA group and C0127+2.5 mg/kg CsA group.The efficacy was evaluated by the following aspects: survival,body weight,GVHD score,serum inflammatory factor level,and pathological section.(3)Transcriptome sequencing(RNA-seq)was performed on the colon tissues of mice in the Control group,aGVHD group,5 mg/kg CsA group,XBJ+2.5 mg/kg CsA group,and C0127+2.5 mg/kg CsA group respectively,and the RNA-seq results were analyzed using the Ingenuity Pathway Analysis(IPA)to obtain the potential mechanisms and targets of XBJ and C0127 protecting the intestinal damage of aGVHD mice.The results were further validated using real-time reverse transcription-polymerase chain reaction(Real-time PCR),Western blot(WB),and immunohistochemistry.Results:(1)Through literature review and learning,it is known that XBJ mainly contains four major categories of compounds: Honghua flavonoids,Chishao monoterpene glycosides,Chuanxiong/Danggui phthalides and Danshen catechols,from which we selected the compounds with the highest content for HPLC detection,and combined into C0127 according to the content of hydroxysafflower yellow A,ferulic acid,paeoniflorin,and protocatechuic aldehyde in XBJ.(2)The results of in vivo experiments showed that the combination of C0127 and low-dose CsA could prevent and treat aGVHD and protect its intestine,which was mainly reflected in the following aspects:(a)improving the survival rate of aGVHD mice;(b)reducing the weight loss rate of mice;(c)reducing the GVHD score;(d)reducing the expression of inflammatory factors;(e)and reducing the injury degree of liver and colon.(3)The results of transcriptome sequencing(RNA-seq)were analyzed.The threshold value of differentially expressed genes in each group was set as log2 Fold Change≥1 and p-value≤0.05 to obtain the potential targets and related pathways.The results showed that XBJ and C0127 could down-regulate the IL-17 signaling pathway and significantly suppresse the expression of G-CSF in this pathway.These results were verified by Real-time PCR,WB,and immunohistochemistry of mouse intestinal tissue.Conclusion:(1)The combination of low-dose CsA and C0127 can effectively prevent and treat aGVHD mice and play a role in protecting the intestinal tract and other major target organs,and the effect of combined administration is better than that of CsA alone,achieve the effect of reducing toxicity and increasing efficiency,and improve the safety of clinical drugs.(2)The combination of C0127 and low-dose CsA can produce a therapeutic effect and mechanism similar to XBJ,which has the potential and possibility of preventing and treating GVHD.To some extent,it can be regarded as the main pharmacodynamic active ingredient of XBJ.