Efficacy And Molecular Mechanism Of Hydroxyurea In The Treatment Of Atherosclerosis Through Gut Microbiota Based On Clinical Findings

Background and Aim:Atherosclerotic diseases are the number one killer of human health,with high disability and mortality rates,posing a serious threat to people’s health.The pathogenesis of atherosclerosis is complex,and its therapeutic targets are still limited,as well as having the disadvantage of more obvious side effects.For instance,statins present adverse effects such as abnormal liver enzymes,rhabdomyolysis and cognitive decline;anti-platelet drugs inevitably increase the risk of bleeding,etc.Therefore,the exploration of new targets for anti-atherosclerotic drugs and the development of novel drugs for them are still very important scientific issues.Recently,we clinically diagnosed a patient of acute coronary syndrome combined with primary thrombocythemia,and the patient was then treated with oral hydroxyurea for primary thrombocythemia for more than two years,after which the patient’s review showed a significant improvement of his coronary atherosclerosis.This led to a keen interest in the potential anti-atherosclerotic pharmacological effects of hydroxyurea,a traditional antiproliferative drug.The aim of this thesis was to confirm the therapeutic effect of oral hydroxyurea on atherosclerosis at the animal level and to explore its possible mechanisms.Since the gut microbiota has been shown to significantly affect lipid metabolism and the development of atherosclerosis,this thesis provides insight into the pharmacological mechanisms of hydroxyurea for the treatment of atherosclerosis by altering the gut microbiota and cholesterol metabolism using microbial genomics and untargeted metabolomics.Methods:Part I:Clinical findings on the anti-atherogenic potential of hydroxyureaTo describe the diagnosis and treatment of a patient with acute coronary syndrome with primary thrombocytosis.Part II:Pharmacological efficacy of hydroxyurea in ameliorating atherosclerosis in Apo E^-/-mice（1）Establishment of the atherosclerosis model of Apo E^-/-mice combined with high cholesterol diet and administration:Apo E^-/-mice were fed a high fat diet containing1.25%cholesterol for 12 weeks to establish the atherosclerosis model,and drug administration was started after the 4th week of modeling in the following groups:I Control group:C57BL/6J mice were given oral saline;II Model group（Model）:Apo E^-/-mice+high-fat diet,oral normal saline;III Low-dose hydroxyurea treatment group（HU-L）:Apo E^-/-mice+high fat diet,hydroxyurea 5 mg/kg/d;IV Medium-dose hydroxyurea treatment group（HU-M）:Apo E^-/-mice+high fat diet,hydroxyurea 10mg/kg/d;V High-dose hydroxyurea treatment group（HU-H）:Apo E^-/-mice+high-fat diet,hydroxyurea 20 mg/kg/d;VI Drug combination treatment group（D+HU）:Apo E^-/-mice+high fat diet,aspirin 5 mg/kg/d,clopidogrel bisulfate 25 mg/kg/d,hydroxyurea20 mg/kg/day;VII Positive drug treatment group（D+S）:aspirin 5 mg/kg/d,clopidogrel bisulfate 25 mg/kg/d and atorvastatin calcium 5 mg/kg/d;（2）Atherosclerosis severity assessment:animal samples were collected for aortic vascular oil red O staining,aortic root H&E staining and aortic root oil red O staining.Corrected plaque area of mice in each group was measured.（3）Determination of plasma pharmacokinetic parameters of oral hydroxyurea:After establishing the determination method of hydroxyurea by liquid chromatography mass spectrometry（LC-MS/MS）,the pharmacokinetic studies of single oral administration of hydroxyurea and multiple oral administration of hydroxyurea were conducted,and the pharmacokinetic related indexes were calculated;（4）Determination of serum arteriosclerosis related indexes:Fasting blood glucose,triglyceride,total cholesterol,low density lipoprotein cholesterol（LDL-C）,oxidized low density lipoprotein cholesterol（ox-LDL）,interleukin-6（IL-6）,interleukin-1β（IL-1β）,tumor necrosis factor-alpha（TNF-ɑ）and other serum indicators related to atherosclerosis were determined using commercial kits.（5）Determination of drug therapy safety indexes;Serum aspartate aminotransferase（AST）,serum alanine aminotransferase（ALT）,serum creatinine（Cre）and other safety indexes were determined using commercial kits.Part III:Study on the mechanism of hydroxyurea in the treatment of Apo E^-/-mice atherosclerosis by improving the gut microbiota and cholesterol absorption。（1）Experimental animals and their grouping are the same as in Part II;（2）Biodiversity analysis of 16S r RNA of the gut microbiota:the steps include the extraction and detection of fecal microbiota DNA,data quality control and OTU basic analysis,data analysis and calculation,and the expression and difference analysis of the gut microbiota of samples and the prediction analysis of the gut microbiota function of samples;（3）Fecal metabolomics analysis:including sample preparation of GC-MS,metabolomics analysis based on GC-MS,data pretreatment and analysis,identification of differential metabolites and level difference analysis;（4）Immunohistochemical staining:small intestinal epithelial sections were prepared,H&E staining and Niemann-Pick C1-like 1protein immunohistochemical staining of small intestinal epithelial.Results:Part I:Clinical findings on the antiatherogenic potential of hydroxyureaHydroxyurea may have antiatherogenic effect.However,further systematic studies are needed,including the confirmation of efficacy and mechanism at the animal model level.Part II:Pharmacological efficacy of hydroxyurea in ameliorating atherosclerosis in Apo E^-/-mice（1）Apo E gene knockout combined with high-fat diet can significantly induce atherosclerosis,and oral hydroxyurea can effectively treat atherosclerosis in model animals;（2）Oral hydroxyurea can significantly reduce the abnormal increase of serum total cholesterol and LDL-C in model animals caused by modeling,thus explaining its pharmacological effect on anti-atherosclerosis to a certain extent.The mechanism of lowering cholesterol needs further analysis;（3）Oral hydroxyurea did not affect the increase of fasting blood glucose and triglyceride in model animals caused by modeling;（4）Oral hydroxyurea did not significantly affect the levels of inflammatory factors significantly related to atherosclerosis in serum of model animals,including IL-1β,IL-6 and TNF-ɑ;（5）Oral hydroxyurea for 8 weeks did not significantly affect the liver and kidney function of model animals,showing good safety;Part III:Study on the mechanism of hydroxyurea in the treatment of Apo E^-/-mice atherosclerosis model by improving the gut microbiota and cholesterol absorption.（1）Oral administration of hydroxyurea significantly affected the gut microbiota in the animal model:fecal microbial genomics showed that oral administration of hydroxyurea significantly improved the disturbed gut microbiota in the Apo E^-/-mouse mice model,corrected the Firmicutes/Bacteroides ratio,increased the abundance of genera negatively associated with atherosclerosis,decreased the abundance of genera positively associated with atherosclerosis,and normalized the disturbed gut microbiota in the model animals.（2）Oral administration of hydroxyurea is capable of treating atherosclerosis by inhibiting cholesterol absorption and lowering circulating cholesterol:fecal metabolomics analysis showed that oral administration of hydroxyurea increased the abundance of fecal cholesterol,stearic acid,and palmitic acid,suggesting a decrease in substances associated with high-fat dietary sources.The mechanism was associated with a decrease in the small intestinal epithelial cholesterol transporter,NPC1L1,by hydroxyurea.（3）Oral hydroxyurea was associated with changes in the gut microbiota and changes in NPC1L1:Correlation analysis between the gut microbiota and NPC1L1content showed that Lachnospiraceae＿UCG-008 and Roseburia were negatively correlated with NPC1L1 expression.Conclusions:（1）Hydroxyurea has therapeutic effect on atherosclerosis caused by Apo E gene knockout mice combined with high-fat diet,and can significantly reduce the total aortic plaque area and aortic root plaque area of mice.（2）Oral administration of hydroxyurea significantly reduced total cholesterol and low-density lipoprotein cholesterol levels in the serum of Apo E^-/-mice combined with high-fat diet,but had no significant effect on fasting glucose and serum inflammatory factors（IL-1β,IL-6,TNF-ɑ）levels.（3）Hydroxyurea can significantly change the gut microbiota of the Apo E^-/-mouse model of atherosclerosis,increasing the abundance of the genus associated with improved atherosclerosis.（4）Oral administration of hydroxyurea significantly reduced the expression of NPC1L1 in the small intestinal epithelium and significantly enriched fecal cholesterol in atherosclerosis model mice,suggesting that oral administration of hydroxyurea achieves pharmacological effects for the treatment of atherosclerosis by inhibiting intestinal absorption of cholesterol.（5）Oral administration of hydroxyurea increased the abundance of Roseburia and Lachnospiraceae＿UCG-008 genus and negatively correlated with the expression of NPC1L1,suggesting that hydroxyurea may alter the expression of NPC1L1 and thus inhibit cholesterol absorption and improve atherosclerosis through the regulation of gut microbiota.（6）In conclusion,the antiatherosclerotic efficacy of hydroxyurea may be mediated through the pharmacological mechanism of regulating intestinal flora and thus inhibiting intestinal absorption of cholesterol.