Study On The Protective Effect And Mechanism Of Fuzi Lizhong Pill On Intestinal Toxicity Of Irinotecan

Objective:In this study,the anti-inflammatory and antidiarrheal effects and molecular biological mechanisms of Fuzi Lizhong Pill were observed and studied through the IκB/NF-κB pathway by establishing a rat model of irinotecan（CPT-11）inflammatory diarrhea,using Fuzi Lizhong Pill as an intervention drug.It provides a new strategy and a certain theoretical basis for clinical alleviation of the limiting toxic effects of irinotecan.Methods:Fifty-six Wistar male rats were randomly divided into control group,model group,berberine group,IKK-βinhibitor group,the low,medium and high dose groups of Fuzi Lizhong Pill according to body weight,with 8 rats in each group.Except for the control group,the remaining rats were injected with irinotecan solution at 125 mg·kg^-1body weight for 5days,and the control rats were intraperitoneally injected with the same dose of normal saline.After successful modeling,the rats in each group were treated with the corresponding drugs for 30 days,and the rats in the control group were gavaged with the same amount of normal saline.Then,the histopathological changes of the colon of rats were observed by hematoxylin-eosin staining（HE staining）.Transmission electron microscopy（TEM）observed the changes of various ultrastructure in the colonic tissues of rats in each group.Enzyme-linked immunosorbent assay（ELISA）measured interleukin-1β（IL-1β）,interleukin-18（IL-18）,cyclooxygenase-2（COX-2）,tumor necrosis factor-α（TNF-α）,transforming growth factor-β1（TGF-β1）,interleukin-4（IL-4）,interleukin-10（IL-10）.Real-time fluorescence quantitative polymerase chain reactin（q RT-PCR）detected the relative expression of NF-κB subunits p65 and p50,IKK-β,NLRP-3,Caspase-1,GSDMD,ASC m RNA in the colon tissues of rats.Western blot（WB）detected the relative expression of NF-κB subunits p65 and p50,IKK-β,NLRP-3,Caspase-1 and ASC proteins in the colon tissues of rats.Results:（1）HE staining showed that the intestinal mucosa of the rat colon tissue in the model group was disorganized compared with the control group,the crypt was missing,the epithelial cells were reduced,and the inflammatory cells had a large number of infiltrates,and the colon tissue of the baberine group,IKK-βinhibitor group,the low,medium and high dose groups of Fuzi Lizhong Pill were improved to varying degrees compared with the colon tissues of rats in the model group.（2）Transmission electron microscopy showed that the intestinal mucosal epithelial cells in the model group were relatively damaged compared with the control group,the cell membrane was locally damaged,and the organelles were significantly swollen,large-scale degradation and absence of microvilli.But the berberine group,IKK-βinhibitor group,the low,medium and high dose groups of Fuzi Lizhong Pill had different degrees of improvement compared with the model group.（3）ELISA method can conclude that the content of IL-1β,IL-18,TGF-β1,COX-2 and TNF-αin rat colon tissue in the model group was higher than that in the control group,and the difference was statistically significant（P<0.01）,and the berberine group,IKK-βinhibitor group,the low,medium and high dose groups of Fuzi Lizhong Pill were reduced to varying degrees compared with the model group.The differences were statistically significant（P<0.05或P<0.01）.While the contents of IL-4 and IL-10 in the colon tissues of rats in the model group were lower than those in the control group（P<0.01）,and the baberine group,IKK-βinhibitor group,the low,medium and high dose groups of Fuzi Lizhong Pill were increased to different degrees compared with the model group,and the differences were statistically significant（P<0.01）.（4）q RT-PCR showed that the expression levels of p65,p50,IKK-β,NLRP-3,Caspase-1,GSDMD,and ASC m RNA in rat colon tissues in the model group were higher than those in the control group,and the differences were statistically significant（P<0.05或P<0.01）,and the berberine group,IKK-βinhibitor group,the low,medium and high dose groups of Fuzi Lizhong Pill were reduced to varying degrees of m RNA expression levels compared with the model group.The difference was statistically significant（P<0.05或P<0.01）.（5）The Western blot method showed that the protein expression levels of p65,p50,IKK-β,NLRP-3,Caspase-1,and ASC in rat colon tissue in the model group were significantly higher than those in the control group,and the difference was statistically significant（P<0.05或P<0.01）.Compared with the model group,the protein expression levels of p65,p50,IKK-β,NLRP-3,Caspase-1,and ASC in the of berberine group,IKK-βinhibitor group,the low,medium and high dose groups of Fuzi Lizhong Pill were reduced to different degrees,and the differences were statistically significant（P<0.05或P<0.01）.Conclusion:Fuzi Lizhong Pill can improve the intestinal mucosal structure of irinotecan-induced inflammatory diarrhea rats,which may be related to its mechanism of reducing the content of pro-inflammatory factors,increasing the content of anti-inflammatory factors,and downregulating p65 and p50 and IKK-βof the IκB/NF-κB pathway.