Mechanism of Qingjie Fuzheng Granules on Alleviating 5-FU Chemotherapy-Induced Intes-tinal Mucositis in Mouse Model of Colorectal Cancer By Regulating Cathepsin B/NLRP3 Pathway
Objective To explore the therapeutic effect and underlying mechanism of Qingjie Fuzheng Granules(QFG)on in-testinal mucositis caused by 5-fluorouracil(5-FU)chemotherapy in mice with colorectal carcinoma based on the Cathepsin B/NL-RP3 pathway.Methods A total of twenty-four male BALB/c mice were selected for subcutaneous inoculation of CT26 cells in the axilla of the right forelimb to construct a subcutaneous graft mouse model of colorectal carcinoma.When the tumor volume grew to 100-300 mm3,the mice were randomly divided into control group,5-FU group,and 5-FU+Qingjie Fuzheng Granule group(5-FU+QFG group),with 8 mice in each group.The control group was given saline gavage and intraperitoneal injection,and mice in the oth-er groups received intraperitoneal injection of 5-FU(50 mg/kg·d-1),once daily for 5 days;the 5-FU+QFG group was given Qingjie Fuzheng Granules(1.0 g/kg·d-1)by gavage during chemotherapy,once daily for 7 days.Mice were observed daily for diarrhea,and their body mass and tumor volume were recorded.After the last administration on the 7th day,the mice were fasted for 12 hours,and then sampled and tested.The histopathological changes in the jejunum were observed by hematoxylin-eosin staining(HE);the expression levels of diamine oxidase(DAO),D-lactic acid(D-LA),endotoxin(ET),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),interleukin-6(IL-6)were measured by enzyme-linked immunosorbent assay(ELISA);the positive expression of Cathepsin B in the jejunal tissue was detected by immunohistochemistry;the relative expression level of tight junction protein ZO-1 and Occlu-din mRNA in jejunal tissue were determined by real-time quantitative polymerase chain reaction(RT-qPCR),and the relative expres-sion level of ZO-1,Occludin,Cathepsin B,NLRP3,ASC,and cleaved Caspase-1 protein in the jejunal tissue were determined by the Western blot method.Results(1)General condition of mice:During the experimental period,QFG significantly reduced the diarrhea symptoms caused by 5-FU chemotherapy(P<0.05),had no significant improvement on body weight loss caused by 5-FU chemotherapy(P>0.05),and had no significant synergistic effect on tumor growth inhibition by 5-FU(P>0.05).(2)Histopathologi-cal changes of jejunum:QFG significantly improved the symptoms of intestinal mucosal injury such as shortening of intestinal mucosal villi,deepening of crypts and inflammatory cell infiltration caused by 5-FU chemotherapy.(3)Serum DAO,D-LA,ET,and TNF-α,IL-1β,IL-6 content:Compared with the 5-FU group,QFG significantly reduced the serum content of DAO,D-LA,E-T,and pro-inflammatory cytokines TNF-α,IL-1β,IL-6 in the serum of mice with colon cancer(P<0.05).(4)Protein expression levels of Cathepsin B,NLRP3,ASC,and cleaved Caspase-1 in jejunal tissue:Compared with the 5-FU group,QFG significantly reduced the relative protein expression of Cathepsin B(P<0.05)and reduced the relative protein expression of NLRP3,ASC and cleaved Cas-pase-1 in jejunal tissue(P<0.05).(5)ZO-1 and Occludin mRNA transcription levels and protein expression levels in jejunal tis-sue:Compared with the 5-FU group,QFG significantly increased the mRNA and relative protein expressions of tight junction pro-teins ZO-1,Occludin in the 5-FU+QFG group(P<0.05).Conclusion By regulating Cathepsin B/NLRP3 signaling pathway,redu-cing proinflammatory cytokine release,inhibiting intestinal mucosal inflammatory responses,and repairing intestinal mucosal barrier damage,Qingjie Fuzheng Granules can alleviate chemotherapeutic intestinal mucositis caused by 5-FU.
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