Protective Effect Of Flavonoids In Propolis Against Lead Toxicity In Mice And Its Mechanism

Lead is a toxic and non-biodegradable heavy metal element,and is considered to be one of the major threats to the environment and human health because of its wide application in industries.Long-term exposure to lead can adversely affect nervous,blood,digestive systems,and eventually lead to serious diseases.Therefore,the treatment of lead poisoning has been a hot topic for scientists.At present,dimercaptosuccinic acid（DMSA）and 2,3-dimercaptopropanesulfonic acid（DMPS）with low toxicity are mainly used in the treatment of severe lead poisoning,and nature substances are recommended for treatment or prevention moderate and mild lead poisoning.Therefore,it is particularly important to seek for the natural product or medicine with physiological benefits.Flavonoids are secondary metabolites of plants that widely exist in nature,which have strong anti-oxidant,anti-inflammatory and immune-enhancing medicinal effects.Propolis is a natural product used in health food,and has a variety of biological activities because it contains flavonoids as its main active ingredient.In this work,to make full use of propolis,the common flavonoids in propolis were taken as the main research object,and flavonoids with strong antioxidant activity were screened through density functional theory and experiment analysis.And the flavonoids with strong antioxidant activity as representative to explore their preventive protective effects and mechanisms on lead-induced damage in liver and kidney tissues,as well as regulation of gut microbiota disorders.In addition,propolis was selected as an applied research to study its ethanol extract’s preventive protective effect on lead-induced mice.The main research contents are as follows:1.NBO analysis,dissociation energy,and conceptual DFT were used to study the abilities of common flavonoids in propolis to chelate metal ions and scavenge free radicals.The results showed that H atoms of the catechol hydroxyl in myricetin,quercetin and luteolin molecules had multiple positive charge distributions and a little dissociation energy,which could be easily attacked by free radicals.In addition,the molecules possessed lower molecular orbital energy gaps and more negative charges distributed on O atoms of hydroxyl,which were conducive to reacting with metal ions（M）and forming stable M-O bonds.2.Spectral characterization and determination of antioxidant activity were used to study the antioxidant activities and complexation effects of myricetin,quercetin and luteolin.The results showed that the HOMO of myricetin molecules had good dispersibility throughout the molecular system,which was conducive to occuring the L→M charge transfer,leading to red-shifts 52 nm in the UV-Vis spectrum.According to the CDA analysis,myricetin molecules can provide 0.549 electrons to the empty orbits of Pb（Ⅱ）to form stable Pb-O bonds,which made the FT-IR spectrum show strongν（Pb-O）at 729 cm^-1.In addition,results of FRAP,ABTS,and DPPH showed that myricetin had strong reducing ability and free radical scavenging ability,among which the DPPH scavenging ability was as high as90.11%.All in all,myricetin has strong antioxidant activity and may become an effective ingredient for removing lead from the body,providing a theoretical basis for further research on the protective effect of flavonoids on lead-exposed mice.3.The preventive protective effect and mechanism of myricetin on lead-induced kidney injury in mice was studied by analyzing changes in metal ion levels,oxidative stress parameters,expression levels of inflammatory cytokines,pathological examination and expression of NF-κB p65 positive cell of epithelial cells in kidney tissue.The results showed that myricetin could effectively prevent the increase of lead content（p<0.05）and the decrease of Calcium,Iron and Zinc levels;and inhibit the increase of BUN and CRE levels in serum;meanwhile,it could effectively reduce the MDA content,and increase the SOD activity and GSH content（p<0.05）;In addition,myricetin significantly down-regulated the expression of inflammatory cytokines by inhibiting the NF-κB pathway-mediated inflammatory response.It could prevent glomerular atrophy and reduce the swelling and hyperemia of the renal tubules.4.The preventive protective effect and mechanism of myricetin on lead-induced liver injury in mice were studied by measuring the metal ion,oxidative stress parameters,inflammatory cytokines and pathological examination of liver and observing the expression of NF-κB p65 positive cell in hepatic epithelial cells by immunohistochemical staining.The results showed that myricetin can effectively prevent the increase of the lead content（p<0.05）and the decrease of metal ions levels in liver tissue of lead-exposed mice.Myricetin effectively inhibited the increase of ALT,AST in serum（p<0.05）and the production of excess MDA,and increased the levels of endogenous antioxidants SOD and GSH（p<0.05）.In addition,myricetin can significantly down-regulate the high expression of TNF-αby inhibiting the inflammatory response mediated by the NF-κB pathway.Mice administered with 100 mg/kg myricetin before lead exposure can prevent damages such as heparin disorders and disordered hepatocyte alignment.5.To explore the regulatory effect of myricetin on the intestinal flora in lead-exposed mice,the microbial community of the intestinal contents of mice were analyzed using 16s rRNA sequencing.The results showed that lead altered the diversity and abundance of gut microbiota in mice,while myricetin effectively prevent the increase of the relative abundance of Mucispirillum and Candidatus＿Arthromitus,and increased the relative abundance of Adlercreutzia at the genus level.Therefore,myricetin can effectively regulate the abundance and diversity of gut microbiota of lead-exposed mice.6.Taking propolis ethanol extract as the research object.Y-maze experiments and oxidative stress were used to explore the preventive protective effects of propolis ethanol extract on lead-induced tissue damage in mice,and flavonoids in its ethanol extract were analyzed by HPLC-DAD.The results showed that the propolis sample contained rutin,myricetin,morin hydrate,luteolin,kaempferol,apigenin,chrysin and galangin.Under lead exposure,preventive intervention of propolis ethanol extract could effectively reduce lead levels in liver,kidney and brain tissues of lead-exposed mice（p<0.05）;it also significantly protected the learing and memory abilities of mice and inhibited the effects of lead on the central nervous system.In addition,the ethanol extract can considerably inhibited oxidative damage to liver,kidney and brain tissues（p<0.05）.In short,the research of preventive protective effect of propolis ethanol extract on lead-exposed mice provides a theoretical basis for the further development of propolis,and also has certain reference value for further research on natural health foods or drugs to prevent lead poisoning in the body.