The Role Of Novel NAC Carbonized Polymer Dots In The Treatment Of Non-alcoholic Fatty Liver Combined With Periodontitis In Mice

Non-alcoholic fatty liver disease（NAFLD）is closely associated with periodontitis,as both diseases share common pathological features such as excessive generation of reactive oxygen species（ROS）and chronic low-grade inflammation.Liver is the main target organ for ROS attack and can promote metabolic inflammation,such as hepatocyte steatosis,insulin resistance,activation of inflammatory signals,and tissue injury,which exacerbates the progression of NAFLD,increases the risk of periodontitis,and affects the treatment effect of periodontitis in NAFLD patients.Therefore,clearing excessive ROS and alleviating oxidative stress and metabolic inflammation are effective approaches for relieving NAFLD.Currently,there are no approved drugs for treating NAFLD.N-acetylcysteine（NAC）is the most common antioxidant small molecule drug,which,upon entering cells,promotes the synthesis of glutathione and sulfane sulfur species to protect cells from oxidative stress damage.It is a potential drug for the prevention and treatment of NAFLD.However,its poor stability and lack of specificity in distribution limit the application of NAC.Nanomedicine,as a key area of drug innovation,has received extensive attention.Among them,carbonized polymer dots（CPDs）,a new type of carbon-based nanomaterial,possess excellent properties such as small size,free radical scavenging ability,enzyme-like activity,spontaneous fluorescence,and good biocompatibility.These properties make CPDs the preferred nanomaterial for biological applications.In view of the above issues,this study designs novel CPDs based on NAC,and observes their effects on ROS clearance,antioxidant performance,and tissue damage reduction.It also explores the potential mechanisms,providing new theoretical and experimental foundations for the future clinical application of NAC-b CPDs.In the chapter two,NAC-b CPDs were prepared by hydrothermal method using NAC and citric acid as raw materials.The crystalline structure of NAC-b CPDs was clear,with uniform size distribution and an average particle size of 1.82 nm.It’s mainly composed of elements such as C,N,O,S and H,enriched with amino,carboxy,and mercapto groups,with a Zeta potential（-18.93 m V）.KMn O₄ redox,Fenton reaction inhibition and catalase experiments confirmed that NAC-b CPDs have good ROS scavenging ability,especially for hydroxyl radicals.Results of cell biocompatibility testing showed that NAC-b CPDs at concentrations below 400μg/m L had no significant effect on the proliferation activity of human periodontal ligament cells and hepatocytes within 72 hours.Laser confocal microscopy results showed that NAC-b CPDs could be effectively taken up by cells,exhibited blue fluorescence,and overlapped with mitochondria labeled with red fluorescence,indicating that NAC-b CPDs could be located in mitochondria.NAC-b CPDs can effectively reduce the inhibitory level of H₂O₂-induced cell proliferation activity,reduce the levels of total ROS and mitochondrial derived ROS,increase mitochondrial membrane potential,and alleviate cellular oxidative stress damage.In animal studies showed that high or low concentrations of NAC-b CPDs had no significant effect on body weight,blood routine,liver injury indexes and histological changes of relevant important organs in normal mice,which confirmed that NAC-b CPDs had good biosafety in vivo.In order to explore the metabolism and distribution of CPDs in vivo,this study further prepared red NAC-r CPDs by solvothermal method,which have similar size and functional group structure to NAC-b CPDs,with an average particle size of 1.89 nm,but N doping and negative Zeta potential（-45.7±1.25 m V）levels are higher.Small animal fluorescence imaging results showed that NAC-r CPDs could circulate throughout the body through the bloodstream and mainly accumulate in liver,maxilla,and kidney tissues,and were excreted out of the body through urine and feces within 24 hours.By designing control experiments and analyzing the potential mechanism of NAC-b CPDs in ROS scavenging,it was found that it had similar free radical scavenging ability and antioxidant properties as NAC molecules due to the presence of sulfhydryl groups and other sulfur elements.In chapter three,NAC-b CPDs alleviated HFD-induced liver injury in mice with NAFLD.In this study,a mouse model of HFD-induced NAFLD was constructed,and it was found that NAC-b CPDs intervention could effectively inhibit the levels of TG,T-CHO,LDL-C,HDL-C and insulin resistance in NAFLD mice while increasing glucose tolerance and insulin sensitivity,thereby alleviating the level of glycolipid metabolism disorders in NAFLD mice.Furthermore,the intervention of NAC-b CPDs reduced the weight of liver and epididymis adipose tissues,along with reduced liver steatosis,inflammatory cell infiltration,disease activity score and liver function injury-related indicators,thus alleviating liver damage and inhibiting the progression of NAFLD.In chapter four,preliminarily explores the mechanisms by which NAC-b CPDs alleviate liver injury in NAFLD mice.This study confirms that the levels of liver cells apoptosis and the oxidative stress marker MDA in HFD-induced NAFLD mice increase,while the levels of antioxidants such as SOD,and GSH-Px significantly decrease.The expression levels of lipid metabolism-related indexes such as lipid synthesis-related genes Srebp-1c and Pparγand fibrosis-related genes Tgfβ1 and Col1α1,significantly increase,while the expression of mitochondria-related fatty acidβ-oxidation-related genes such as Pparαand Cpt1a significantly decrease.The expression levels of IR-related indexes such as gluconeogenesis-related genes Pepck and G6pase in liver tissues were increased,while glycogen granule deposition was reduced.Inflammatory response indicators in liver tissue,such as CD68⁺macrophage infiltration,TNF-α,TLR4,and NF-κB protein expression levels,significantly increase,accompanied by an increase in systemic inflammatory mediators such as LPS,TNF-α,IL-1β,and IL-6.When NAC-b CPDs were administered,oxidative stress,glycolipid metabolism disorders,inflammatory responses,and TLR4-NF-κB inflammatory signals were inhibited in the liver tissues of NAFLD mice.Thus,NAC-b CPDs can significantly improve the oxidative stress-metabolic inflammatory microenvironment,alleviate liver injury,and reduce the risk of liver fibrosis.In the chapter five,a mouse model of combined NAFLD and periodontitis was induced through silk ligation and high-fat diet feeding.The results of general condition assessments,liver histology,and fecal 16S r RNA sequencing in the mice showed that experimental periodontitis worsened liver injury,disease activity score,and alterations in gut microbiota in NAFLD mice.Micro-CT and histological examinations revealed that HFD promoted alveolar bone resorption,osteoclast formation,and inflammatory cell infiltration in the periodontal tissues of mice with experimental periodontitis,exacerbating periodontal tissue damage.Spearman correlation analysis showed a positive correlation between the extent of liver lesions and alveolar bone resorption levels in mice.However,intervention with NAC-b CPDs effectively alleviated periodontal injury and oxidative stress-metabolic inflammation microenvironment in NAFLD periodontitis mice,demonstrating its potential in treating periodontal tissue damage in NAFLD.In summary,the special metabolic microenvironment of NALFD,namely high oxidative stress-metabolic inflammatory response,was the breakthrough point,and through the regulation of the synthesis method,we successfully prepared two new NAC-b CPDs and NAC-r CPDs,providing new research ideas and experimental evidence for the future application of novel CPDs in NAFLD periodontitis.