Artesunate Alleviates Intestinal Injury Induced By Irinotecan And Enhances Anti-tumor Activity

Objective:Irinotecan is a topoisomerase I inhibitor that was approved in 1994 for the treatment of cancer.To date,a derivative of this natural product remains the world’s leading anti-cancer drug.However,the clinical use of irinotecan is limited due to its side effects,the most concerning of which is the intestinal toxicity caused by irinotecan.Studies have reported that anti-aging is associated with alleviating the side effects of chemotherapy.Irinotecan is believed to have the potential to induce cellular senescence,so it is important to find drugs that are anti-irinotecan to cause aging to alleviate side effects.In addition,irinotecan as an anti-tumor drug with significant therapeutic effect,if the drug can relieve the side effects while synergistic treatment to improve the anti-tumor activity of irinotecan,the anti-tumor effect will be doubled.Artesunate,as a drug with multiple potential therapeutic effects,has significant anti-inflammatory,anti-apoptotic,and anti-tumor pharmacological activities in various disease models.However,its research in the field of anti-aging has not been reported,and it is expected to conduct research on anti-aging and anti-inflammatory effects in aging models.Therefore,this study aims to explore the therapeutic effect of artesunate as a drug with potential multiple therapeutic efficacies in irinotecan-induced aging and intestinal side effects models and analyze its intrinsic mechanism of action through in vitro and in vitro experiments,and to evaluate the synergistic effect of artesunate on irinotecan anti-tumor therapy.Method:1.Establish a cellular senescence model induced by H₂O₂,and detect the expression changes of SA-β-gal and the expression of p16,p21,p53,SASP and other aging-related indicators under the action of artesunate in this model,so as to evaluate the anti-aging effect of artesunate.2.In the irinotecan-induced HUVEC（human umbilical endothelial vein cell）cell senescence model and HIEC（intestinal epithelial）cell senescence model,the beneficial effects of artesunate on cytotoxicity and cell senescence were explored by cck8 cell proliferation detection and aging related index detection.The mechanism of artesunate anti-aging and anti-inflammatory was explored in an irinotecan-induced model of cellular senescence,and Western blot and qRT-PCR were used to detect the expression of pmTOR,p-p65,p-p38 and other proteins and mRNA levels of a range of pro-inflammatory factors.3.An 8-week-old C57BL/6 male mouse was selected to establish an irinotecan-induced intestinal injury and aging model,and the protective effect and anti-aging effect of artesunate on the intestine in vivo were evaluated by monitoring mouse weight,intake and water intake,HE staining,SA-β-gal staining,Western blot and qRT-PCR.4.Using HCT116 colorectal cancer cell line,cck8 cell proliferation and crystal violet staining were used to observe the killing effect of artesunate and irinotecan on tumor cells.5.Finally,8-week-old male mice of BALB/c nude were selected for in vivo xenograft experiments to evaluate the tumor growth rate and volume size of mice,and HE staining and immunohistochemistry detected inflammatory infiltration and Ki-67,PCNA proliferation and BCL2 and caspase3 apoptosis indicators in tumor tissues,and verified the inhibitory effect of drug combination therapy on tumor growth in mice.Results:1.In the H₂O₂-induced HUVEC cell senescence model,we found that artesunate downregulated the expression of SA-β-gal and other aging indexes,and the expression differences between each group were statistically significant（P<0.05）,significantly inhibiting H₂O₂-induced cellular senescence.2.Artesunate significantly inhibited irinotecan-induced aging in HUVEC cells and HIEC cells,and the effect of the optimal anti-aging concentration of artesunate on cell viability remained at about 90%,which was not statistically significant,indicating the safety of this concentration.3.Irinotecan accelerates the aging process in vivo and in vitro,which we believe is mainly caused by activating mTOR signaling,while artesunate inhibits the expression of mTOR and thus blocks the aging process.4.In response to cellular inflammatory processes,irinotecan upregulated the protein expression of p-p65 and p-p38 and the mRNA levels of TNF-α,IL-1βand IL6 had significant statistical significance（P<0.05）,while artesunate treatment inhibited cellular inflammation and downregulated the above indexes,which was statistically significant（P<0.001 compared with irinotecan treatment group）.5.Artesunate treatment reduces the inflammatory infiltration of the colonic ileum of mice by reducing TNF-α,IL-1βand IL6 mRNA levels of intestinal tissue,and prevents irinotecan-induced intestinal injury by reversing weight loss,increasing the intake of water intake in mice,improving colon length,etc.thereby improving irinotecan-induced intestinal injury.6.Irinotecan treatment led to aging of ileum and colon tissues in mice,with a high positive rate of SA-β-gal staining,and activated the protein expression of p16 and p-mTOR in intestinal tissue,which was statistically significant（P<0.001）,mRNA levels of p21 and p53also increased（P<0.05）,and the combination treatment of artesunate and irinotecan blocked the intestinal aging process and lowered a series of aging indicators.7.In HCT116 cells,the combination treatment of irinotecan and artesunate significantly enhanced the killing effect on tumor cells.The cell viability of 3μM irinotecan+6μM artesunate treatment was reduced to about 20%,which was statistically significant compared with the control group（P<0.001）,and also downregulated the protein expression of BCL2 and promoted apoptosis of tumor cells.8.In the in vivo xenograft tumor model,artesunate and irinotecan combination therapy increased the expression of caspase3 in tumor tissues,reduced the expression of BCL2 and induced apoptosis of tumor cells,while inhibiting the levels of Ki-67 and PCNA to reduce the proliferation of tumor cells,and the expression differences between the groups were statistically significant（P<0.05）.The results of histological analysis showed that the combination therapy restored the tissue structure arrangement disorder in the control group and alleviated the inflammatory infiltrate in the tumor tissue.Conclusion:Artesunate has a variety of beneficial pharmacological activities,which can not only effectively inhibit the aging caused by irinotecan by regulating the expression of phosphorylated mTOR,downregulate the expression of cellular inflammation,and even reduce the intestinal toxicity caused by irinotecan,inhibit intestinal aging,and enhance the antitumor effect of irinotecan.Therefore,artesunate is expected to be developed as a potential adjuvant drug for anti-tumor therapy to prevent or alleviate the adverse reactions of irinotecan in clinical treatment and improve the success rate of tumor treatment.