Effects Of Haze Exposure On Inflammation,oxidation And Apoptosis Of Peripheral Blood Of Indoor And Outdoor Workers And Mechanism Of Amelioration Of PM2.5 Induced Lung Injury By IL-10

In recent years,with the acceleration of urban development and modernization,air pollution is becoming more and more serious,and its impact on human health has become a major research topic.Air pollutants include gaseous pollutants and particulate matter(particle matters,PM).PM pathogenicity depends on its size,composition,source,solubility and ability to produce reactive oxygen species.Studies have shown that smoke is usually caused by high concentrations of fine particles(particle size less than or equal to 2.5 btm,or PM2.5)or aerosols.Studies have found that the PM2.5 small in diameter has a large surface area,so it can carry various toxic substances and destroy other parts of the body.More importantly,the incidence of respiratory diseases is increased.Shijiazhuang in China is one of the top ten polluted cities in the world.The average PM2.5 concentration in Shijiazhuang from April2014 to April 2015 was 101μg/m~3,10.1 times higher than the World Health Organization air quality guidelines(10μg/m3).More worrying is that air pollution is classified as I carcinogens.In recent years,more and more studies in toxicology,epidemiology and other related fields have shown that respirable particulate matter is closely related to the incidence and mortality.As one of the factors leading to non-accidental human death,PM2.5 are closely related to adverse health consequences,and the underlying mechanisms of its impact on human health include oxidative stress,inflammatory response,and apoptosis.Our study is divided into two parts:the first part is the clinical part,Compared with a wide range of animal and cell studies on haze in vitro,human studies may be more representative and persuasive.First,we detected the atmospheric level of the PM2.5,and then we measured the index of IL-6,IL-10 and SOD in terms of oxidative stress and inflammation.To further understand the deep effects of PM2.5 on the body,we also detected the apoptosis rate of lymphocytes and mitochondrial membrane potential in circulation.The second part is the animal experiment part.We established a PM2.5 acute exposure model in rat by dropping PM2.5 suspension into the rat trachea.To understand the lung injury,we detected HE staining of lung tissue.The subcell structrures including mitochondria were observed under electron microscope.Meanwhile,cytokines and antioxidant enzymes were detected.To combat the haze we chose IL-10 to antagonize lung injury.Subcutaneous injection of IL-10 was given to rats,and the effects of IL-10 on lung injury induced by PM2.5 were evaluated from the aspects of inflammation,oxidation and apoptosis including AMPK/SIRT1/PGC-1αsignaling pathway.Part One Effects of Haze Exposure on Inflammation,Oxidation and Apoptosis Indicators of Peripheral Blood of Indoor and Outdoor WorkersObjective:To observe the levels of IL-6,IL-10,SOD,apoptosis rate of lymphocytes and mitochondrial membrane potential in peripheral blood of indoor and outdoor workers exposure to haze,and evaluate the health effects of haze on indoor and outdoor workers.Mehtods:The subjects were divided into two groups,From September 1,2020 to September 30,2020,the outdoor group selected from the construction site workers of cardiovascular ward of Hebei Provincial people’s Hospital of Shijiazhuang City as the research object,and the indoor group selected from the physical examination personnel of physical examination center of Hebei Provincial people’s Hospital.Concentrations of indoor and outdoor PM2.5were monitored.The serum of all the observed individuals was collected.SOD enzyme activity was detected by self-oxidation of benzotriol,and the levels of IL-6(interleukin-6,IL-6)as proinflammatory factor and IL-10(interleukin-10,IL-10)as anti-inflammatory factor were measured by enzyme-linked immunosorbent assay.Apoptosis in peripheral blood cells was detected by flow cytometry FITC-Annexin V/PI double staining.Rho 123 method was used to detect mitochondrial membrane potential.Results:1.General data of patients enrolled:We recruited a total of 90 males divided into two groups.The age of the outdoor group fluctuated from 26～63years old with an average(46±12)years old,and the age of the indoor group fluctuated from 25 to 62 years old with an average(44±11)years old.Differences of gender and age between the two groups were not statistically significant(P>0.05).2.Result of atmospheric PM 2.5 index:individual PM2.5 exposure concentrations in indoor group were 38.23(27.03,49.25)μg/m~3,and individual PM2.5 exposure concentrations in outdoor group were60.64(34.58,86.59)μg/m~3.Differences of exposure concentrations between the two groups were statistically significant(P<0.05).3.Inflammation and oxidation index:the SOD,IL-10 level of indoor group was higher than that of outdoor group,and the IL-6 level was lower than that of outdoor group.The difference between the two groups was significant(P<0.05).4.The apoptosis index:the apoptosis rate of peripheral blood lymphocytes in indoor group was 1.64(1.10,2.32)%,which was significantly lower than that in outdoor group 3.32(2.21,4.31)%.The apoptosis rate between the two groups was statistically significant(Z value=-4.075,P=0.000,P<0.05).5.Mitochondrial membrane potential:the mitochondrial membrane potential of indoor group was 40.00(29.10,53.30)%,which was significantly higher than that of outdoor group 34.30(27.65,40.47)%.The mitochondrial membrane potential of the two groups was statistically significant(Z value-2.240,P=0.025,P<0.05).Summary:In the study of 90 indoor and outdoor workers,PM2.5interfered with the individual immune defense response,and induced oxidative stress and apoptosis.1.PM 2.5 increased the IL-6 level,while reduced the IL-10 level.PM 2.5induced inflammatory reaction and interfered with the indivdual immune defense response.2.PM2.5 reduced the SOD level in the circulation,which lead to oxidative stress.3.PM2.5 reduced the mitochondrial membrane potential of peripheral blood lymphocytes,and increased the apoptosis rate of lymphocytes.PM 2.5induced apoptosis.Part Two IL-10 ameliorates PM2.5-induced lung injury by AMPK/SIRT1/PGC-1αpathwayObjective:This study investigated the protective effect of IL-10 against lung injury and the possible involvement of AMPK/SIRT1/PGC-1αsignaling.Methods:1.Animals and exposure regimen:The rats were randomly allocated to five groups(eight rats/group):(1)saline control group;(2)-(4)low-,mid-,and high-dose PM2.5 groups;and(5)high-dose PM2.5+recombinant rat IL-10(rr IL-10)group.The rats in the control group were injected with 300μl saline10 times(every other day).The rats in the low-,mid-,and high-dose PM2.5groups were treated 10 times(every other day)with low(1.8 mg/kg bw),medium(5.4 mg/kg bw),or high(16.2 mg/kg bw)doses of PM2.5 suspension,respectively,by carefully inserting a straight gavage needle(12 G)into the trachea via the rima glottidis after anesthetization.The rats in the PM2.5+rr IL-10 group were administered high-dose PM2.5 10 times(every other day),followed 1 min later by subcutaneous injection of 5μg rr IL-10.2.Measurement indexes:The mean diameter,particle size distribution and Zeta potential of PM2.5 samples were evaluated by a Zetasizer Nano ZS90 analyzer.Histopathological changes of lung were observed by hematoxylin-eosin staining after modeling.The ultrastructural changes of lung tissue were evaluated by transmission electron microscopy.Lung apoptosis was detected by TUNEL.Inflammatory factors in rat alveolar lavage fluid was determination by enzyme linked immunoassay(ELISA),MDA and GSH-Px test kits for GSH-Px of oxidase and antioxidant enzymes in lung tissue.To investigate whether the AMPK/SIRT1/PGC-1αaxis plays a role in the protection of PM2.5 toxicity,The expression of AMPK,SIRT1 and PGC-1αproteins and m RNA was detected by western blotting method.3.Statistical analysis:SPSS 21.0 statistical software was used for the statistical analysis.Statistical differences between multiple groups were analyzed by one-way analysis of variance(ANOVA).P<0.05 was considered statistically significant.Results:1.PM2.5 physicochemical characterization:The Zetasizer Nano ZS90analyzer showed that the mean PM2.5 size collected in a non-industrial district in winter was 1287.67±217.01 nm.The polymer dispersity index of the PM2.5suspensions varied from 0.378 to 0.439,indicating homogeneous dispersion.The mean zeta potential of the PM2.5 suspensions was-27.85±0.32 mv,indicating good stability.In summary,the PM2.5 samples exhibited super miniature size,good uniformity,and good stability.The mass concentrations of seven heavy metals in the PM2.5 samples,comprising carcinogenic metals(As,Cd,Cr,and Ni)and non-carcinogenic metals(Pb,Hg,and Mn).As338.61μg/g,Cd 19.58μg/g,Cr 87.02μg/g,Ni 30.20μg/g,Pb 187.32μg/g,Hg 0.85μg/g,Mn 230.58μg/g.2.HE staining of rat lung tissue:The control rats had generally normal lung morphology.The lung tissues of the rats in the low-dose PM2.5 group exhibited thickening of the alveolar septa with lymphocyte infiltration.In the mid-dose PM2.5 group,large areas of inflammatory cell infiltration were observed around the bronchial walls.Furthermore,in the high-dose PM2.5group,there was widespread lung inflammation,represented by massive infiltration of lymphocytes and macrophages that destroyed the airway and alveolar structures,with black particles deposited in the alveolar areas.Additionally,there was exfoliation of luminal epithelial cells.In contrast,lung inflammation was alleviated in the PM2.5+rr IL-10 group.3.Ultrastructural injury of lung in rats under electron microscope:In the control group,normal lamellar bodies were observed in the type II alveolar epithelial cells,and the structures of the alveolar epithelial cells,including the mitochondria,were by and large intact.However,after PM2.5 treatment,the perinuclear space was increased,and the structures of the alveolar epithelial cells and the mitochondria were destroyed.The lamellar bodies in the type II alveolar epithelial cells displayed vacuolation,and the mitochondria appeared swollen.In particular,in the high-dose PM2.5-treated rats,mitochondria vacuolation became highly obvious,some of the mitochondrial cristae disappeared,and degranulation of the rough endoplasmic reticulum(loss of ribosomes)was observed.In comparison to in the high-dose PM2.5-treated groups,the deterioration of the type II alveolar epithelial cells and mitochondria was significantly reduced by rr IL-10.4.IL-10 reduces PM2.5-induced inflammatory cytokine production in the BALF.A dose-dependent increase in TNF-αwas observed in the three PM2.5groups compared to the control group(P<0.05).The IL-10 and IL-22 levels in the BALF were also markedly higher in the middle-and high-dose PM2.5groups than in the control group(P<0.05).However,the IL-10 level in the high-dose PM2.5 group showed a sharp decline compared to in the mid-dose PM2.5 group(P<0.05).Importantly,compared to the high-dose PM2.5 group,the PM2.5+rr IL-10 group exhibited dramatically decreased TNF-αand IL-22levels and an increased IL-10 level(P<0.05).5.IL-10 attenuates PM2.5-induced oxidative stress in the lungs.Compared to the control group,MDA concentrations in the mid-and high-dose groups significantly increased(P<0.05).GSH-Px activity dramatically and dose dependently decreased in the three PM2.5 groups compared to the control group(P<0.05).Treatment with rr IL-10 led to a pronounced decrease in the MDA level,and a pronounced increase in the GSH-Px activity,compared to in the high-dose PM2.5 group(P<0.05).These results indicate that rr IL-10 reduces the oxidative stress induced by PM2.5.6.IL-10 attenuates PM2.5-induced apoptosis in the lungs.TUNEL analysis was used to detect apoptosis of the lung cells.There was only a small proportion of apoptotic cells in the saline control group,but this proportion gradually increased with the PM2.5 dose,along with increased staining intensity of apoptotic nuclei.Consequently,the apoptosis index(AI)in each of the groups was consistent with the TUNEL staining intensity.The AI was consistently significantly higher in the PM2.5-treated rats than the control rats(p<0.05).However,rr IL-10 treatment significantly decreased the AI in the PM2.5+rr IL-10 rats compared to the high-dose PM2.5 rats(P<0.05).7.PM2.5downregulates,whileIL-10upregulates,AMPK/SIRT1/PGC-1αsignaling.Toexaminewhetherthe AMPK/SIRT1/PGC-1αaxis plays a role in the protective effects of IL-10against PM2.5-induced toxicity,the p-AMPK,AMPK,SIRT1,and PGC-1αprotein expression was assessed by western blotting.The results showed that compared to in the control group,the protein levels of p-AMPK,SIRT1,and PGC-1α,along with the p-AMPK to AMPK ratio,were dramatically downregulated by PM2.5 in a dose-dependent manner(all p<0.05).As expected,after treatment with IL-10,the protein levels of p-AMPK,SIRT1,and PGC-1αand the p-AMPK to AMPK ratio were significantly increased compared to the levels in the high-dose PM2.5 group.The trends regarding AMPK,SIRT1,and PGC-1αm RNA expression were similar to those regarding the protein expression.These results indicate that IL-10 protects lung tissues against PM2.5 toxicity,which might involve AMPK/SIRT1/PGC-1αsignaling-mediated mitochondrial biogenesis.Summary:1.PM2.5 downregulates AMPK,SIRT1,and PGC-1αexpre ssion.2.IL-10 significantly attenuates oxidative stress and apoptosis.3.IL-10 reduces PM2.5-induced injury by downregulating AMPK/SIRT1/PGC-1αsignaling.Conclusions:Our results demonstrate that IL-10 has protective effects against PM2.5-induced pulmonary injury by reducing inflammatory responses,oxidative stress,and apoptosis via upregulating AMPK/SIRT1/PGC-1αsignaling.We propose that the amelioration of mitochondrial dysfunction by IL-10 is significant regarding the control of inflammation.This study indicates that IL-10 may be developed as a therapeutic intervention for alleviating PM2.5-induced inflammation,and that the AMPK/SIRT1/PGC-1αpathway may provide potential targets for additional future treatments.