Protective Effect And Mechanism Of Andrographolide On 5-Fu Induced Intestinal Mucositis

5-Fluorouracil(5-Fu)is the most frequently prescribed anti-tumor drug for the treatment of various types of cancer,including gastrointestinal,breast,head and neck carcinomas.About 2 million patients are annually treated with 5-Fu worldwide.As an anti-metabolite drug,5-Fu selectively killed the tumor cells and rapidly proliferating cells(e.g.intestinal cells),and more than 50%of cancer patients receiving 5-Fu chemotherapy suffered from intestinal mucositis,which caused problems such as neutropenia,malnutrition,dysbiosis,and even systemic infection and septicemia risk.Intestinal mucositis causes problems with prognosis,severely affects the quality of life in most cancer patients.Thus,it is necessary to clarify the mechanism of 5-Fu-induced intestinal mucositis and to find effective drugs against it.Andrographis paniculata is a common traditional Chinese medicine;the channel tropism is heart,lung,large intestine,and bladder with effect of clearing away heat and detoxification,cooling blood and reducing swelling.Usually Andrographis panicata is suitable for cold and fever,sore mouth and tongue,diarrhea and dysentery,heat drenching and astringent pain,etc.Andrographolide(Andro)is one of the main effective components of Andrographis panicata.In recent decades,Andro has been extensively used for its different therapeutic properties,including anti-inflammatory and anti-viral activities.Many studies have shown that Andro and its analogs can alleviate diarrhea caused by colitis,senna or castor oil.However,no studies have reported whether Andro can alleviate 5-Fu induced intestinal mucositis.The purpose of this study was to establish a suitable model of 5-Fu-induced intestinal injury in mice and study the mechanism of intestinal injury.Based on the model,we will explore the protective effect and potential molecular mechanism of Andro on 5-Fu-related intestinal injury.It lays a theoretical foundation for the clinical application of Andro in 5-Fu-related intestinal injury.Part I:Assessment of dose-response relationship of 5-fluorouracil to murine intestinal injuryAims:This study aimed to determine the proper dose of 5-Fu in the murine model,and assess the dose-response relationship between the severity of intestinal injury and doses of 5-Fu.Main methods:42 Male BALB/C mice were divided into 6 groups to observe the effects of doses of 5-Fu(25,50,100,200,400 mg/kg/d)on intestinal damage in mice.(1)The weight change,diarrhea and survival status of mice were observed to assess the effect of 5-Fu in mice macroscopically.(2)Hematoxylin-eosin staining was used to investigate the pathological changes(including villus height and crypt depth)of ileum and colon in mice after 5-Fu treatment.(3)We tested the level of diamine oxidase in plasma of mice by ELISA to investigate the degree of micro-damage of intestinal mucosa in mice challenged by 5-Fu.(4)Cellular apoptosis was determined in ileum and colon tissues by terminal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL)assay.(5)Western blot was used to detect the expression of Bax,Bcl-2 and caspase-3 related proteins in ileum and colon.Results:The weight of mice in 5-Fu groups were decreased gradually with the time of administration of 5-Fu.At the same time,more severe diarrhea and higher mortality were observed with the increase of 5-Fu dosage.Histopathological damage was more severe in mice that received higher doses of 5-Fu.In addition,plasma diamine oxidase(DAO)activity of 5-Fu treated mice decreased with intestinal injury in a dose-dependent way.TUNEL and western blot analysis showed that cell apoptosis in the ileum and colon were also related to 5-Fu dosage.However,administration of 200 and 400 mg/kg 5-Fu resulted in extremely high mortality,severe diarrhea rate and histopathological damage,but 25 mg/kg 5-Fu did not cause significant intestinal damage.Western blot analysis showed that 5-Fu induced intestinal loss may be related to the up-regulation of Bax and caspase 3 and down-regulation of Bcl-2.Conclusions:The severity of 5-Fu related intestinal injury was dose-dependent.To ensure the appropriate pathological damage and mortality of experimental mice,the proper dosage of 5-Fu to induce proper intestinal mucositis was 50 mg/kg/d to 100 mg/kg/d.Part Ⅱ Protective effect of Andrographolide on 5-Fu induced intestinal mucositis by regulating p38 MAPK signaling pathwayAims:The present study aimed to investigate the effect of Andro on the 5-Fu induced intestinal mucositis and the potential mechanism.Main methods:Male BALB/C Mice model was injected 100mg/kg 5-Fu for 5 days to establish intestinal mucositis.Andro at different doses(25,50,100mg/kg/day)were given orally.Weight loss,diarrhea score and mortality were recorded.Hematoxylin-eosin staining was used to investigate the pathological changes(including villus height and crypt depth)of ileum and colon in mice after intervened by Andro in 5-Fu-challenged mice.The cell apoptosis and proliferation were evaluated by TUNEL and PCNA assay respectively.Apoptosis related proteins including p38,p53,Bax/Bcl-2,caspase8/3 were detected by Western blot to explore the possible mechanism.Results:Andro significantly relieved 5-Fu induced weight loss and diarrhea in a dose-dependent manner in vivo.HE assay showed that the histopathological damage,including villus height ang crypt depth,was attenuated by the Andro.TUNEL assay showed that Andro decreased the percentage of TUNEL-positive intestinal cells as compared to the 5-Fu group,while the PCNA assay showed that the PCNA positive cells were increased significantly after Andro treatment compared with 5-Fu group.Besides,Andro obviously down-regulated the protein expression of caspase3 and caspase8,inhibited the phosphoration of p38 and p53 which were intensified by the 5-Fu treatment,and downregulated the Bax expression.Conclusions:Andro significantly attenuated the intestinal damage induced by 5-Fu,and improved the weight loss and diarrhea of mice.This effect was related to the inhibition of p3 8 MAPK pathway.Part Ⅲ:In vitro verification of andrographolide in alleviating intestinal injury induced by 5-Fu through p38 pathwayAims:To investigate the effect of Andro on 5-Fu-induced intestinal injury in NCM460 cells in vitro,and to explore the role of p38 MAPK pathway.Methods:NCM460 cells challenged by 5-Fu was treated with different doses of Andro to investigate the effect of Andro on 5-Fu-induced apoptosis.At the same time,we used the p38 inhibitor SB203580 and p38 agonist asiatic acid to demonstrate the mechanism of Andro on 5-Fu-induced intestinal toxicity.CCK-8 was used to determine the cell proliferation,flow cytometry was used to analyze the apoptosis,and Western blot was used to detect the expression of p38 and other apoptosis related proteins.Results:5-Fu significantly increased the apoptosis of NCM460 cells in a dose-dependent manner,which was significantly inhibited by Andro use.At the same time,the application of 5-Fu led to a significant decrease in p38 and p53 phosphorylation,reduced the expression of cleaved caspase 8/3,up-regulated the Bax and down-regulated of Bcl-2 expression in NCM460 cells.At the same time,flow cytometry showed that SB203580 played a protective effect on 5-Fu-induced cell damage,while the application of p38 activator asiatic acid partially reversed the protective effect of Andro on 5-Fu-related cell damage;Western blot also confirmed that asiatic acid could partially reverse the effect of Andro on the expression of apoptosis related protein.Conclusion:These results suggested that up regulation of p38MAPK was an important mechanism of 5-Fu-induced intestinal injury,and Andro can protect the cell from 5-Fu-induced apoptosis,and then played a protective role in 5-Fu-related intestinal injury by inhibit the phosphorylation of p38MAPK.Part Ⅳ:The effect of andrographolide on the pharmacokinetics and pharmacodynamics of 5-Fu in miceObjective:To investigate the effect of Andro on the pharmacokinetics and the antitumor effect of 5-Fu.Methods:A HPLC-MS/MS method for the determination of 5-Fu in mouse plasma was established.280 mice were randomly divided into 4 groups:single dose of 5-Fu group;Andro+5-Fu single dose group;multi dose use of 5-Fu group;Andro+5-Fu multi-dose combination group.Blood samples were collected according to time points and the pharmacokinetic parameters of 5-Fu were calculated.Another 24 mice were inoculated with H22 cells in the abdominal cavity to establish the transplanted tumor model.The mice were randomly divided into four groups:control group,5-Fu group,Andro group and 5-Fu+Andro group.The weight changes and tumor weight of the mice were recorded.Results:The linear range of 5-Fu in mice plasma was 0.05-100 μg/mL,and the RSD value of intra assay and inter assay precision was less than 15%.The pharmacokinetic analysis showed that Andro didn’t affected the pharmacokinetic parameters of 5-Fu.Compared with the control group,5-Fu alone and Andro could significantly inhibit the growth of H22 transplanted tumor in mice.Andro 100 mg/kg combined with 5-Fu could enhance the antitumor effect of 5-Fu in tumor bearing mice,but there was no significant difference compared with 5-Fu alone.Conclusion:Andro does not affect the pharmacokinetics of 5-Fu in mice,nor does it affect the antitumor effect of 5-Fu in tumor bearing mice.