Study On The Protective Effects And Mechanisms Of Sulforaphane Against PM2.5-Induced HBE Cell Injury And Mouse Lung Injury

Particulate matter 2.5（PM2.5）is a mixture of airborne solid particles and liquid droplets with an aerodynamic diameter of 2.5 microns or less.In recent years,PM2.5 has been a public issue of concern as it continues to pose a threat to human health.Due to the complex composition of PM2.5 and the large spatial and temporal variability,there are still limitations in the understanding of the mechanisms of PM2.5 toxicity.Nevertheless,there is an emerging consensus that oxidative stress and inflammatory responses play a critical role in triggering and exacerbating PM2.5-related chronic diseases.Given the need of economic development,it is unlikely to control PM2.5 at sources in a short period of time.At the same time,PM2.5 can still cause damages to human tissues and organs through cumulative effects at low concentrations and under chronic exposure conditions,and there is no"safe concentration"for PM2.5 exposure.Therefore,in addition to policy interventions,physical filtration and pharmacological treatments,new and effective interventions are urgently needed to prevent or mitigate the health effects of PM2.5.In recent years,the results from in vitro and in vivo models and population studies have continued to show that nutritional or dietary interventions offer new ideas to attenuate the impacts of PM2.5 on public health:mainly through the intake of one or more bioactive substances with antioxidant or anti-inflammatory or immunomodulatory properties,which can prevent or reduce the negative effects of PM2.5 on human health.Sulforaphane is an excellent natural phytochemical with a variety of physiological activities including antioxidant,anti-inflammatory and anti-cancer.It is a potential and excellent bioactive substance for achieving nutritional interventions.However,the effect of sulforaphane on human health damage caused by PM2.5 and its related mechanisms are still unknown.Therefore,in this study,sulforaphane was selected as the phytochemical.After collecting and characterizing PM2.5 samples,in vitro and in vivo models regarding PM2.5 damages were constructed to investigate the effect of sulforaphane on PM2.5-induced damages in in vitro and in vivo models.Meanwhile,the protective effects of sulforaphane and its underlying mechanisms were investigated in combination with multi-omics studies.The main findings of this study are as follows:（1）The Air Metrics Mini Vol Portable Sampler and Wuhan Tianhong TH-1000H were used to collect airborne particulate matter.The average particle size is 338.13 nm,which belongs to the PM2.5 category.Under SEM,PM2.5 aerosol particles appear irregularly shaped.Under TEM,PM2.5 aerosol particles appear in aggregates and clusters（carbon aggregates）,mainly from motor vehicle emissions and biomass combustion near the sampling site.Among the PM2.5 surface elements,the most abundant was C,which accounted for 58.86%of the total elemental content（except Si and O）.A total of 25 elements,including K,Fe,Na and Ca,were detected in the PM2.5 samples,the majority of which originated from the earth’s crust,with Zn being the most abundant element among the anthropogenic sources,accounting for 8.55%.The PM2.5 samples collected contained 15 PAH substances in the order of acenaphthene,acenaphthene,dibenzo（a,h）anthracene,pyrene,benzo（b）fluoranthene,benzo（a）anthracene,benzo（k）fluoranthene,poly（fluorene）,benzo（G,H,I）perylene,benzo（a）pyrene,pyrene,fluoranthene,naphthalene,phenanthrene and anthracene.Among them,acenaphthene and acenaphthene were the most abundant,occupying 49.65%of the total PAH content.Meanwhile,four ions,NH₄⁺,Cl^-,SO₄^2-and NO₃^-,were detected in the aqueous solution of PM2.5 samples,in the order of SO₄^2-（70.46±0.59μg/m³）,NH₄⁺（18.95±1.63μg/m³）,Cl^-（8.58±0.34μg/m³）and NO₃^-（65.30±1.83μg/m³）.The ratio of NO₃^-to SO₄^2-was close to 1,and motor vehicle emissions were the main source of NO₃^-and SO₄^2-in PM2.5 in the sampling area.（2）An in vitro model of oxidative damage to HBE cells by PM2.5 was constructed.In this model,the effects of PM2.5 and sulforaphane on HBE cells and potential mechanisms were explored in terms of cell viability,lactate dehydrogenase release,malondialdehyde content,intracellular reactive oxygen species levels,apoptosis rate and inflammatory factors.The results showed that PM2.5 exposure significantly increased the level of reactive oxygen species in HBE cells,disrupting the integrity of the cell membrane and leading to the release of intracellular lactate dehydrogenase into the culture medium,and the excessive production of reactive oxygen species reacted with lipids to increase malondialdehyde content.The expressions of IL-6 and IL-8 are increased.Oxidative stress and inflammatory response are the main molecular mechanisms of PM2.5 damage.The above physiological and biochemical parameters were improved by the intervention of sulforaphane,indicating that the intervention of sulforaphane protected the HBE cells from oxidative damages caused by PM2.5.The protective mechanism of sulforaphane was investigated by enzyme-linked immunoblotting.The results showed that sulforaphane treatment significantly enhanced the Nrf2-Keap1signaling pathway,promoted the binding of Nrf2 into the nucleus with antioxidant response elements,increased the transcriptional level of antioxidant genes and prompted an increase in the expression of key downstream antioxidant proteases.The enhanced Nrf2-Keap1 signaling pathway,which compensates for the oxidative stress caused by PM2.5 in HBE cells,may be one of the molecular mechanisms by which sulforaphane alleviates PM2.5 damage to HBE cells.（3）An in vivo model of PM2.5 damage in mice was constructed,and the protective effect of the intervention with sulforaphane was examined.The mechanism of PM2.5 damage in mice and the mechanism of the interventional effect of sulforaphane are described in conjunction with multi-omics analysis.Compared to normal mice,PM2.5 exposure caused significant clinical symptoms in mice without affecting their normal behavioural state.These included an abnormal increase in body weight,a significant increase in body fat,changes in blood markers associated with abnormal lipid metabolism（including increases in TG,TC,LDL-C,AST and ALT,and a decrease in HDL-C）,and increases in lung-to-body,lipid-to-body and liver-to-body ratios.In addition,PM2.5 exposure caused oxidative stress and inflammatory responses in the lungs,while PM2.5 exposure caused systemic low-grade inflammatory responses（mainly increased levels of IL-1β,TNF-αand MCP-1）and immune imbalances of T helper cells in mice.The above clinical symptoms,pathological damage and local and systemic inflammatory responses were significantly improved after sulforaphane intervention.（4）PM2.5 exposure led to disruptions in the intestinal flora of mice,mainly in the form of pathogenic genera including Helicobacter,Candidatus＿Saccharimonas,Desulfovibrio,Lachnoclostridium and other pathogenic genera,which are strongly associated with inflammatory responses,abnormal glucose and lipid metabolism and obesity in the body,have increased in abundance;while the beneficial genera mainly include Dubosiella,Carnobacterium,Rikenella,Turicibacter,Akkermansia,Lachnospiraceae＿NK4A136＿group,Bifidobacterium and others have decreased in abundance.In contrast,sulforaphane administration reversed the dysbiosis,possibly through direct intervention of the intestinal flora and its metabolites to restore flora homeostasis,and possibly indirectly through activation of the Nrf2 pathway to modulate oxidative stress and inflammatory responses in the body.（5）Metabolomic analysis showed that the metabolites that differed between the groups were mainly organic acids and their derivatives,as well as some lipids and lipid-like small molecules;they were mainly involved in the body’s metabolism（including total metabolism,lipid metabolism and amino acid metabolism）and mainly affected the body’s digestive and endocrine systems.In the PC group compared with the NC group,the differential metabolism was mainly enriched in metabolic pathways,pyrimidine metabolism,tryptophan metabolism,etc.It also involved some purine metabolism,protein digestion and absorption,glutathione metabolism,fatty acid biosynthesis,bile secretion,etc.In the SFN-High group compared with the PC group,the differential metabolites were enriched in fatty acid biosynthesis,aldosterone synthesis and secretion,protein digestion and absorption,and amino acid biosynthesis,etc.The results suggest that PM2.5 exposure and sulforaphane intervention are strongly associated with changes in metabolic and inflammatory status in mice.（6）Transcriptomic analysis showed that PM2.5 exposure significantly upregulated the expression levels of genes related to leukocyte migration,trending,phagocytosis and defence against bacterial invasion,cellular antioxidant and insulin regulation in mouse lungs.In the sulforaphane administration intervention group,the reduced inflammatory response and improved oxidative stress status in the lungs resulted in significant down-regulation of gene expression levels related to antioxidant function,chemokine activity,receptor ligand activity and cytokine activity at the gene level in the sulforaphane administration group compared to the PM2.5 exposure group,indicating that the sulforaphane administration intervention enhanced the body’s antioxidant and anti-inflammatory capacity and reduced pulmonary and systemic microinflammatory responses.（7）The activation of the Nrf2-Keap1 pathway and the increased expression of its downstream antioxidant-related proteins（e.g.HO-1）caused by sulforaphane in lung tissues may be one of the mechanisms by which sulforaphane prevents PM2.5 lung injury;the significant increase in the expression level of PPARγcaused by carotenoid intervention in adipose tissues may be one of the mechanisms by which sulforaphane ameliorates the metabolic disorder,microinflammatory response and body weight abnormalities caused by PM2.5 in mice It may be one of the mechanisms by which sulforaphane improves metabolic disorders,microinflammatory responses and body weight abnormalities in mice caused by PM2.5.