Atorvastatin Calcium Alleviates Intestinal Damage Caused By Fluorouracil And Enhances Its Anti-tumor Efficacy

Objective:Human umbilical vein vascular endothelial cells（HUVEC）were treated with H₂O₂ to screen for the anti-aging drug atorvastatin calcium.To understand whether5-fluorouracil causes normal tissue cell senescence,we treated HUVEC and normal human colonic epithelial cells（HIEC）in our experiment.Meanwhile,we also need to understand whether Atorvastatin calcium can alleviate cellular senescence caused by 5-fluorouracil and verify whether Atorvastatin calcium can enhance the anti-tumor activity of 5-fluorouracil in HCT116 cells and animal experiments on BALB/c nude xenografted tumors.Methods:Using HUVEC,a model cell to study senescence,a hydrogen peroxide-induced HUVEC cell aging model was established,and the anti-aging drug Atorvastatin calcium was screened in a library of 474 small molecule compounds approved by the Food and Drug Administration（FDA）.We established the 5-fluorouracil-induced HUVEC and HIEC cell senescence model.During the experiment,SA-β-Gal glycosidase staining was used to detect the percentage of senescent cells;Western blot was used to detect protein expression levels,RT-q PCR detected m RNA expression levels;CCK-8 cytometry detected the effect of drugs on cell viability;Atorvastatin calcium was observed to have an antagonistic effect on cellular senescence caused by 5-fluorouracil.The model of 5-fluorouracil-induced intestinal injury in6-8-week-old male BALB/c mice was successfully constituted and may be helpful to evaluate the impact and mechanism of atorvastatin calcium.Next,Kras-positive HCT116 cancer cells were used to investigate the anti-tumor efficacy of Atorvastatin calcium combination with5-fluorouracil.8-week-old male mice of BALB/c nude were selected to inoculate HCT116tumor cells on the back of their left thigh,and the anti-tumor efficacy of Atorvastatin calcium was observed through treatment.HE staining and immunohistochemistry was used to detect the proliferation index（Ki-67,PCNA）and apoptosis index（Cleaved-caspase-3,Bcl-2）to determine the combined anti-tumor efficacy of atorvastatin calcium.Results:1.In the oxidative stress model of human umbilical vein vascular endothelial cells（HUVEC）,Atorvastatin calcium can alleviate cellular senescence caused by hydrogen peroxide.HUVEC cells were treated continuously with hydrogen peroxide 490μmol/L for 90minutes and incubated at 37°C and 5%CO₂ for 4 days after the exchange of the complete medium to construct an oxidative stress-induced HUVEC cell aging model.After HUVEC treatment for 90 minutes for hydrogen peroxide during the screening of drugs,different concentrations of Atorvastatin calcium were added to the solution.SA-β-Gal glycosidase staining was performed after 4 days of co-incubation.It was found that Atorvastatin calcium could alleviate hydrogen peroxide-induced aging of HUVEC cells.The proportion of SA-β-Gal positive cells in the Atorvastatin calcium group was significantly reduced.On day 3,the m RNA expression level was considerably reduced by aging-related protein p16 and aging-related genes（p53,IL-1β,IL-6,TNF-α）.2.5-fluorouracil can induce the aging of HUVEC and HIEC cells,and Atorvastatin calcium can alleviate normal cell senescence caused by 5-fluorouracilIn the HUVEC and HIEC cell models,we observed that both cells showed morphological enlargement and flattening when 5-fluorouracil was treated,and meanwhile,SA-β-Gal-positive cells increased significantly.After adding Atorvastatin calcium to5-fluorouracil-induced HIEC cells,a statistically significant decrease in SA-β-Gal-positive cells was observed.The expression levels of aging-related proteins and genes were statistically significant.The experimental results show that 5-fluorouracil can cause colonic epithelial cell aging,while Atorvastatin calcium can alleviate 5-fluorouracil-induced aging of HIEC cells.3.In the BALB/c mouse model,Atorvastatin calcium can alleviate the damage caused by5-fluorouracil to the intestine of mice through anti-aging effects.The intestinal injury model of BALBc mice was established by intraperitoneal injection of fluorouracil.We observed that the diarrhoea index,body weight,24-hour food intake,24-hour water intake,histopathological observation,the m RNA expression of colon tissue and p16protein expression index,the above results all showed that the intestines of mice in the5-fluorouracil group were damaged to a certain extent,which was related to the aging of intestinal epithelial cells caused by 5-fluorouracil.Atorvastatin calcium can alleviate enteritis caused by 5-fluorouracil,and this repair function is closely associated with the anti-aging effect of atorvastatin calcium.4.Mechanism research about Atorvastatin calcium alleviating 5-fluorouracil-induced aging of human colonic epithelial cells4.1 In vitro,Atorvastatin calcium alleviates human colonic epithelial cell aging caused by5-fluorouracil Dependence on the m TOR signaling pathway.In human colonic epithelial cells（HUVEC cells）,1μmol/L of 5-fluorouracil has been identified as the lowest concentration of 5-fluorouracil-induced HIEC senescence.Our study also found that on 1 and 3 days,5-fluorouracil gradually activated m TOR signaling pathways.In contrast,m TOR protein expression levels were significantly progressively reduced with the combination of atorvastatin calcium.When m TOR activator insulin was used,m TOR protein expression increased again,demonstrating the 5-fluorouracil-dependent fight against aging by activating the m TOR signaling pathway.4.2 In a mouse model of BALB/c diarrhoea,Atorvastatin calcium relies on the m TOR signaling pathway to alleviate intestinal damage caused by 5-fluorouracil.Firstly,we collected mouse colon tissue proteins.Western blot experiments proved that5-fluorouracil activated the m TOR signaling pathway in intestinal damage,However,Atorvastatin calcium could alleviate the damage caused by 5-fluorouracil to the mouse intestine by inhibiting the m TOR signaling pathway.5.In vivo and in vitro,Atorvastatin calcium relieves intestinal damage caused by5-fluorouracil relying on the p65 NF-κB and p38MAPK anti-inflammatory pathway.5-fluorouracil could activate the levels of phosphorylation pathway of inflammation-related proteins p65 and p38,while,Atorvastatin calcium could be reversed,reducing the protein phosphorylation levels of p65 and p38 and also decreasing the expression of m RNA levels of inflammatory factors IL-1β,IL-6 and TNF-αmediated by the p65 NF-κB/p38MAPK signaling pathway,and alleviated intestinal inflammation caused by 5-fluorouracil.In the BALB/c mouse model,Atorvastatin calcium significantly reduced the protein phosphorylation level of p65 and p38 and the expression level of m RNA of downstream inflammatory factors（IL-1β,IL-6 TNF-α）.These results prove that Atorvastatin calcium can reduce intestinal damage caused by 5-fluorouracil by relieving intestinal inflammation.6.Atorvastatin calcium enhances 5-fluorouracil sensitivity in HCT116 cells and BALB/c nude mouse tumor models.6.1 In vitro,Atorvastatin calcium enhances the anti-tumor efficacy of 5-fluorouracil on HCT116 cells.Cell viability of HCT116 was measured using CCK-8 on days 2 and 3,respectively.When HCT116 cells were treated with different concentrations of 5-fluorouracil alone,the survival rate of HCT116 cells treated with 8μmol/L 5-fluorouracil on the 2^nd day was more than 80%;The survival rate of HCT116 cells treated with 8μmol/L 5-fluorouracil on 3^rd day is about 60%;However,when Atorvastatin calcium combined with 5-fluorouracil treated HCT116 cells,the combined anti-tumor effect increased significantly followed by the increase of Atorvastatin calcium concentration.the cell survival rate of Atorvastatin calcium5μmol/L combined with 5-fluorouracil 8μmol/L on day 2 was close to 0;the mortality rate of tumor cells of 1μmol/L,3μmol/L and 5μmol/L Atorvastatin calcium combined with 5μmol/L5-fluorouracil was less than 50%,respectively.The mortality rate was nearly 0 when combined with 8μmol/L of 5-fluorouracil.the above results show that Atorvastatin calcium enhances the anticancer sensitivity of 5-fluorouracil.6.2 Tumor treatment effect of Atorvastatin calcium enhancing 5-fluorouracil on BALB/c nude miceBy subcutaneous injection of HCT116 cells on the left thigh of BALB/c nude mice,anti-tumor treatment was carried out after the tumor volume of the mice reached 100mm³,starting from inoculation with HCT116,closely observing and measuring the size of the tumor until the tumor in the control group increased to 1000-2000mm³,the mice were sacrificed,the tumor tissue was isolated,and paraffin embedding fixation was performed.HE and immunohistochemical assays are performed.It was found that compared with the control group,5-fluorouracil can reduce tumor to some extent,but the tumor volume and weight of BALB/c nude mice in the Atorvastatin calcium combined with the 5-fluorouracil group were significantly smaller than those in fluorouracil alone.The results of the proliferation index Ki-67,PCNA,and apoptosis indicators（Cleaved-caspase-3,Bcl-2）in immunohistochemistry also showed that the anti-tumor effect of Atorvastatin calcium combined with 5-fluorouracil was significantly better than that of monotherapy.It was preliminarily assessed according to the body weight change curves every day during treatment,indicating that Atorvastatin calcium could be toleranced to mice.The treatment was within a safe range,with no mice dying due to treatment.Conclusion:5-fluorouracil is the most widely used first-line chemotherapy drug in patients with advanced colorectal cancer.Still,its adverse side effects,especially moderate to severe diarrhoea,are among the leading causes of anti-tumor failure.The clinical need for treatment to reduce intestinal damage caused by 5-fluorouracil has yet to be met;we studied the relationship between Atorvastatin calcium and 5-fluorouracil-induced intestinal injury in a BALB/c mouse model.This study observed that Atorvastatin calcium could inhibit5-fluorouracil-induced cellular senescence in a HIEC model.Atorvastatin calcium significantly alleviated intestinal damage by inhibiting senescent cell accumulation and intestinal inflammation in animal models.Similarly,the intestinal protective effect of Atorvastatin calcium is associated with aging and inflammation.At the same time,administering Atorvastatin calcium during 5-fluorouracil therapy will suppress tumor development.In addition,we observed that this effect of Atorvastatin calcium was closely related to the inhibition of the m TOR signaling pathway in5-fluorouracil-treated cells and BALB/c mice.In the BALB/c nude mouse model,we determined that Atorvastatin calcium improved the anti-tumor sensitivity of 5-fluorouracil.Atorvastatin calcium is expected to be developed into a new anti-tumor drug that reduces intestinal damage in patients to meet clinical needs and provides a new treatment strategy and theoretical basis for the selection and medication of clinical cancer patients.